Name: functional assessment of kinesin-7 cenp-e in spermatocytes using in vivo inhibition, immunofluorescence and flow cytometry
Uploaded: 2021-12-28T14:00:00
Description: Watch this Scientific Journal Video about Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry at JoVE.com

We thank all members of the Cytoskeleton Laboratory at Fujian Medical University for helpful discussions. We thank Jun-Jin Lin at Public Technology Service Center, Fujian Medical University for technical assistances in flow cytometry. We thank Ming-Xia Wu and Lin-Ying Zhou at Electron Microscopy Lab of Public Technology Service Center, Fujian Medical University for technical assistances in electron microscopy. We thank Si-Yi Zheng, Ying Lin, Qi Ke, and Jun Song at Experimental Teaching Center of Basic Medical Sciences at Fujian Medical University for their supports. This study was supported by the following grants: National Natural Science Foundation of China (grant number 82001608), Natural Science Foundation of Fujian Province, China (grant number 2019J05071), Fujian Provincial Health Technology Project (grant number 2018-1-69), Startup Fund for Scientific Research, Fujian Medical University (grant number 2017XQ1001), Fujian Medical University high level talents scientific research start-up funding project (grant number XRCZX2017025) and Research project of online education and teaching of Chinese medicine graduate students (grant number B-YXC20200202-06).

All animal experiments were reviewed and approved by the Animal Care and Use Committee at Fujian Medical University (Protocol number SYXK 2016-0007). All mouse experiments were performed in accordance with the relevant guidelines of the Care and Use of Laboratory Animals of the National Institutes of Health (NIH publications number 8023, revised 1978).
1. Construction of GSK923295-mediated CENP-E inhibition mouse models
<ol>
	<li>Sterilize the surgical instruments at&#160;12...

In this study, we have established an in vivo CENP-E inhibition model of mouse testes using the abdominal surgery and microinjection of GSK923295. The abdominal surgery and testicular injection method used in this study has the following advantages. First, it is not limited to the age of mice. Experimenters can perform testicular injection at an early stage, for example, at 3-week-old or younger mice. Second, GSK923295 has a specific and excellent inhibitory effect on CENP-E. Third, this method is simple to oper...

Meiosis is one of the most important, highly rigid, evolutionary conserved events in eukaryotic organisms, and is essential for gametogenesis, sexual reproduction, genome integrity, and genetic diversity1,2,3. In mammals, the germ cells undergo two successive cell divisions, meiosis I and II, after a single round of DNA replication. Unlike sister chromatids in mitosis, duplicated homologous chromosomes pair up and segregate into two daughter cells during meiosis I4,5. In meiosis II, sister chromatids pull apart and segregate to form haploid gametes without DNA replication6. Mistakes in either of the two meiotic divisions, including spindle assembly defects and chromosome missegregation, can result in the loss of gametes, sterility or aneuploidy syndromes7,8,9.
Accumulating studies have shown that kinesin family motors play a crucial role in the regulation of chromosome alignment and segregation, spindle assembly, cytokinesis, and cell cycle progression in both mitotic and meiotic cells10,11,12. Kinesin-7 CENP-E (Centromere protein E) is a plus-end-directed kinetochore motor required for chromosome congression, chromosome transport and alignment, and the regulation of spindle assembly checkpoint in mitosis13,14,15,16,17,18. During meiosis, CENP-E inhibition by the specific inhibitor GSK923295 leads to cell cycle arrest, chromosome misalignment, spindle disorganization, and genome instability in spermatogenic cells19. The localization patterns and dynamics of CENP-E at the centromeres of dividing spermatocytes indicate that CENP-E interacts with kinetochore proteins for the sequential assembly of centromeres during meiosis I20,21. In oocytes, CENP-E is required for chromosome alignment and the completion of meiosis I13,22,23. Antibodies or morpholino injection of CENP-E results in misaligned chromosomes, abnormal kinetochore orientation, and meiosis I arrest in both mouse and Drosophila oocytes23. Compared with the essential roles of CENP-E in mitosis, the functions and mechanisms of CENP-E in meiosis remain largely unknown. Detailed mechanisms of CENP-E in chromosome congression and genome stability in male meiotic cells remain to be clarified.
Spermatogenesis is a complex and long-lasting physiology process, involving sequential spermatogonia proliferation, meiosis and spermiogenesis. Therefore, the whole process is extraordinarily difficult to be reproduced in vitro in mammals and other species24,25. It is impossible to induce spermatocytes differentiation after the pachytene stage in vitro. Studies on male meiotic divisions have been generally limited to experimental analyses of early meiotic prophase25,26. Despite many technological endeavors, including short-term culture of spermatocytes27,28 and organ culture methods25, there are few effective methods to study male meiotic division. Furthermore, genetic deletion of essential genes usually results in developmental arrest and embryonic lethality. For example, mouse embryos lacking CENP-E fail to implant and cannot develop past implantation29, which is an obstacle in mechanistic studies of CENP-E in meiosis. Taken together, establishing a practical and feasible system to study male meiotic division can greatly promote the research field of meiosis.
The small cell-permeable inhibitor is a powerful tool to study kinesin motors in cell division and developmental processes. The allosteric inhibitor, GSK923295, specifically binds to CENP-E motor domain, blocks the release of ADP (adenosine diphosphate), and finally stabilizes the interactions between CENP-E and microtubules30. In this study, an in vivo inhibition mouse model is presented through abdominal surgery and testicular injection of GSK923295. CENP-E inhibition results in chromosome misalignment in metaphase I of primary spermatocytes. Furthermore, CENP-E inhibition leads to meiotic arrest of spermatocytes and the disruption of spermatogenesis. A series of protocols are described for the analyses of spermatocytes and can be applied to observe meiotic spindle microtubules, homologous chromosomes, and subcellular organelles in spermatocytes. Our in vivo inhibition method is an effective method for the studies of meiotic division and spermatogenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td>Sterile.</td>
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		<tr>
			<td>1.5 mL Centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm Petri dish</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
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			<td>95% Ethanol</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10009164</td>
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			<td>tubulin rabbit polyclonal antibody</td>
			<td>Beyotime</td>
			<td>AF0001</td>
			<td>For immunofluorescence assays. Use at 1:100.</td>
		</tr>
		<tr>
			<td>rabbit anti-Histone H3 (phospho S10) monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab267372</td>
			<td>For immunofluorescence assays. Use at 1:100.</td>
		</tr>
		<tr>
			<td>rabbit anti-TUBA4A polyclonal antibody</td>
			<td>Sangon Biotech</td>
			<td>D110022</td>
			<td>For immunofluorescence assays. Use at 1:100.</td>
		</tr>
		<tr>
			<td>Anti-SYCP3 rabbit monoclonal antibody</td>
			<td>Abcam</td>
			<td>ab175191</td>
			<td>For immunofluorescence assays. Use at 1:100.</td>
		</tr>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>CITOTEST</td>
			<td>188105</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 488-labeled goat anti-rabbit antibody</td>
			<td>Beyotime</td>
			<td>A0423</td>
			<td>Sencodary antibody. Use at 1:500.</td>
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			<td>Aluminium potassium sulphate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10001060</td>
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			<td>Anhydrous ethanol</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>100092690</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fade mounting medium</td>
			<td>Beyotime</td>
			<td>P0131</td>
			<td>Prevent photobleching of flourescent signals.</td>
		</tr>
		<tr>
			<td>BD FACS Canto II</td>
			<td>BD Biosciences</td>
			<td>FACS Canto II</td>
			<td></td>
		</tr>
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			<td>Bovine Serum Albumin</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>69003435</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424BK745380</td>
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		</tr>
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			<td>Chloral hydrate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>80037516</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Shanghai Experiment Reagent Co., Ltd</td>
			<td>122670</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sangon Biotech</td>
			<td>A004194-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>CITOTEST</td>
			<td>10212020C</td>
			<td>20 &#215; 20 mm. Thickness 0.13-0.16 mm.</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Beyotime</td>
			<td>C1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Dye vat</td>
			<td>Several commercial brands available</td>
			<td>91347802</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y, alcohol soluble</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>71014460</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10009318</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde - aqueous solution</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10010018</td>
			<td></td>
		</tr>
		<tr>
			<td>GSK923295</td>
			<td>MedChemExpress</td>
			<td>HY-10299</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin, anhydrous</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>71020784</td>
			<td></td>
		</tr>
		<tr>
			<td>ICR mouse</td>
			<td>Shanghai SLAC Laboratory Animal Co., Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td>Fluorescent image analysis.</td>
		</tr>
		<tr>
			<td>Leica ultramicrotome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica EM UC-7 ultramicrotome</td>
			<td>Leica</td>
			<td>EM UC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Modfit MFLT32</td>
			<td>Verity Software House</td>
			<td></td>
			<td>For analysis of flow cytometry results.</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral gum</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10004160</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti-S2 microscope</td>
			<td>Nikon</td>
			<td>Ti-S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Picric acid</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>J60807</td>
			<td></td>
		</tr>
		<tr>
			<td>Rheodyne</td>
			<td>Sangon Biotech</td>
			<td>F519160-0001</td>
			<td>10 &#956;l rheodyne</td>
		</tr>
		<tr>
			<td>Sliced paraffin</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>69019461</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium iodate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>80117214</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td>For abdominal surgery. Sterilize at 121 &#176;C, 20 min.</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>FEI</td>
			<td>Tecnai G2</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Shanghai Experiment Reagent Co., Ltd</td>
			<td>173970</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>30188928</td>
			<td>Dilute in sterile PBS to make a 0.25% working solution.</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>30189328</td>
			<td>Dilute in sterile PBS to make a 0.1% working solution.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>80096618</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10023418</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional assessment of kinesin-7 cenp-e in spermatocytes using in vivo inhibition, immunofluorescence and flow cytometry

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

We have successfully constructed an in vivo CENP-E inhibition model of mouse testes through abdominal surgery and testicular injection of GSK92329519. The key technical steps of this method were shown in Figure 1. After testicular injection of GSK923295 for 4 days, the testes were harvested for further analyses. In the control group, the spermatogenic wave in the seminiferous tubules was regular and organized (Figure 2A). However...

Watch this Scientific Journal Video about Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry at JoVE.com

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

This article reports an in vivo inhibition of CENP-E through abdominal surgery and testicular injection of GSK923295, a valuable model for male meiotic division. Using the immunofluorescence, flow cytometry and transmission electron microscopy assays, we show that CENP-E inhibition results in chromosome misalignment and genome instability in mouse spermatocytes.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...

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