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>
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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.

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	<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 ...

Aneuploidy is the leading genetic cause of early miscarriage in humans. Most errors in chromosome segregation occur during meiosis in oocytes. Therefore, evaluating the spindle assembly checkpoint in oocytes is critical to understanding why oocytes are error-prone.Here we describe three techniques to comprehensively evaluate SAC integrity in mouse oocytes by examining different critical steps at checkpoint using a combination of live imaging and immunofluorescence. Demonstrating the procedure will be Dr.Cecilia Blengini, a research associate from my laboratory. To begin prepare one milliliter of culture media containing nocodazole and reversine with DMSO as a control as described in the manuscript.For oocyte maturation and live imaging use a pre-warmed 96-well plate in an incubator. And load the first, second and third well with 150 microliters of the control DMSO, nocodazole and nocodazole plus reversine treatment respectively. While keeping the plate in the incubator under the conditions as described in the manuscript.To start myotic maturation, remove milrinone by sequentially transferring the oocytes through 6 drops of 100 microliters of milrinone free culture media containing DMSO. And place them into the corresponding well of the 96-well plate. Using a hand or mouth-operated pipette while viewing the oocytes under a stereo microscope.Image the oocytes using a bright-field microscope equipped with an incubator chamber with a controlled environment at 37 degrees Celsius, 5%carbon dioxide and 80%humidity. Capture images at the middle plane of the oocytes at intervals of 20 minutes for 24 hours. After quantifying the number of oocytes that extrude a polar body by identifying the cells that go through asymmetrical cytokines with a small cell next to the egg and within the shared zona pellucida.View the images using imaging software. Microinject pro-phase one arrested oocytes with 100 nanograms per microliters of previously prepared securin-gfp cRNA. After microinjection, allow the oocytes to recover and translate the RNA in the carbon dioxide incubator for at least three hours.Then load 150 microliters of culture media with or without five micromolar of nocodazole. And 150 microliters of culture media with 0.5 micromolar of reversine in three different wells of a 96-well plate. Wash the microinjected oocytes through six drops of milrinone free culture media And transfer one-third of the oocytes into each treatment.Keep the plate in the incubator at 37 degrees Celsius with 5%carbon dioxide and 80%humidity until the oocytes resume meiosis, around three hours post milrinone washing. Record images of oocyte maturation using a fluorescence microscope equipped with an incubator chamber. Use image analysis software to quantify securin-gfp destruction and open the images of the GFP channel, starting at time 0.1.In the preferred analysis software, use the selection or shape tool to mark each oocyte and generate a region of interest for each cell. Use the same region of interest to select a background area to be subtracted later and measure GFP pixel intensity in each region of interest at all the time points. Lastly to each GFP value, subtract the measurement of the region of interest in the background.After splitting meiosis into three groups, transfer each group of meiosis into 100 microliter drops of milrinone free culture media. Cover the drops with mineral oil and place them in the incubator for three, five, and seven hours to reach the early prometaphase one, late prometaphase one and metaphase one stage respectively. At the assigned time points, fix the oocytes by transferring them into 500 microliters drops of 2%paraformaldehyde in PME buffer for 20 minutes at room temperature using a nine-well glass dish.Then transfer the cells to a clean well containing a 500 microliter drop of blocking solution. To continue MAD2 detection, transfer the oocytes to a clean well containing a 500 microliter drop of permeabilization solution. After incubating for 20 minutes at room temperature, transfer the cells to a new well of blocking solution and incubate for 10 minutes.Transfer the oocytes into a 30 microliter drop of blocking solution with anti-MAD2 antibody and anti-centromere antibody and incubate for two hours at room temperature. To wash excess primary antibodies after transferring the cells to 30 microliters drop of PME buffer supplemented with 0.5%Triton, incubate for 10 minutes in the humidified chamber. Transfer the cells to a 30 microliter drop of blocking solution containing secondary antibodies such as anti-human 633 and anti-rabbit 568.And incubate for one hour at room temperature. To mount the cells on the microscope slide, transfer the cells to a 10 microliter drop of mounting media containing DAPI. Add small dots of petroleum jelly to each corner of the cover slip.Carefully place them on top of the mounting media drop and press slowly to distribute. Seal the cover slip to the slide using clear nail polish. Image kinetochores using a confocal microscope equipped with a 40 or 63 X objective.And using the anti-centromere antibody signal determine the Z range that allows imaging of the entire chromosome area. The DMSO treated control oocytes extruded a polar body around 14 hours after release from milrinone. After SAC activation, nocodazole treated oocytes were arrested in metaphase one and did not extrude a polar body.However, in the absence of SAC activation, oocytes extruded a polar body despite having no kinetochore microtubule attachments. The rate of securin-gfp degradation during myotic maturation was evaluated. Wherein the control DMSO treated oocytes securin-gfp intensity starts to decrease at approximately 10 hours after milrinone washout.When oocytes were matured in the presence of the SAC activating agent, nocodazole, securin-gfp levels were stable during the entire period of myotic maturation. In contrast, when preventing SAC activation with reversine treatment the securin-gfp pattern accelerated and degradation began at approximately four hours after milrinone washout consistent with SAC inhibition. Confocal imaging was employed to detect centromeres and MAD2 of oocytes matured to early prometaphase one, late prometaphase one and metaphase one.To evaluate the strength of the SAC signal, the kinetochore localized, MAD2 pixel intensity was quantified. Significant levels of MAD2 recruitment to kinetochores occur in early prometaphase one when most kinetochores were not attached to microtubules. The levels of MAD2 reduced in later prometaphase and were nearly absent at metaphase one when all kinetochores were stably attached to microtubules.It's important to highlight that each of these techniques have their limitations and their interpretation. And we suggest performing a combination of these methods to evaluate the SAC integrity. These three essays allows researchers to evaluate the stringency of the sac in oocytes giving insight about a potential cause of aneuploidy in human eggs.

Research

JoVE Journal

Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

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Live Cell Imaging of Chromosome Segregation During Mitosis

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An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

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