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.

evaluation-spindle-assembly-checkpoint-integrity-mouse

Research

JoVE Journal

Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

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

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

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

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...