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;121 &#176;C for 30 min. Irradiate the surgical ultra-clean workbench with ultraviolet-C (UVC)&#160;for 2 h. Weigh the 3-week-old male ICR (Institute of Cancer Research) mice used for the experiments and calculate the doses of anesthetic required.</li>
	<li>Anesthetize mice by administering a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection. Check for the depth of anesthesia of the mice through a combination of mouse corneal reflex, nociceptive reflex, respiration, as well as muscle tone. Confirm the mice are deeply anesthetized. 
	NOTE: Place the animal on a heating pad to provide thermal support&#160;during the surgery.</li>
	<li>Tie up the mouse limbs and fix them on the wax tray. Place a drop of vet ointment on mouse eyes to prevent dryness while under anesthesia. Shave the abdominal hair of the mouse from the lower abdomen to the scrotum using an experimental animal razor.&#160;Secure the surgical area with a sterile drape.</li>
	<li>Disinfect the ventral abdomen with a Betadine scrub followed by 75% ethanol&#160;three times. Open the abdominal cavity using a sterile scalpel and make a &#60;5 mm opening.</li>
	<li>Clamp the skin with sterile surgical clamps and pull the epididymal fat pad with sterile dissecting forceps to locate the testes using a sterile tweezers. Fix the testis with sterile forceps under a stereoscope, and slowly inject 10 &#181;L of GSK923295 into seminiferous tubules at a final concentration of 10 &#181;M using 10 &#181;L of rheodyne30. For the construction of the control group, inject 10 &#181;L of 1% DMSO (dimethyl sulfoxide)/PBS (phosphate buffered saline) solution. 
	NOTE: Store the GSK923295 solution at a concentration of 10 mM at -80 &#176;C. Dissolve 0.1 &#181;L of 10 mM GSK923295 into 100 &#181;L of PBS solution to prepare the 10 &#181;M of GSK923295 solution.</li>
	<li>Gently push the testis back into the abdominal cavity with sterile surgical forceps. Suture the peritoneum and skin separately with two to four stitches using the suture line with a diameter of 0.1 mm.</li>
	<li>Label a 3 x 3 mm place on the back of the animal with a permanent marker after abdominal surgery, put the mouse back to the feeding cage and ensure a clean and pathogen-free environment with sufficient food and water.</li>
	<li>Keep the environment in a sterile state through the filtered air, the sterilized food and water. Take care of the animal until it has regained sufficient consciousness to maintain sternal recumbency. For post-operative analgesia, administer a dose of buprenorphine (0.1 mg/kg) subcutaneously every 12 h for 3 days. Ensure the mouse is not returned to the company of other animals until fully recovered. 
	NOTE: The mice are given postoperative care, and appropriately give the wound local anesthesia using 0.5% lidocaine to reduce postoperative pain when necessary.</li>
</ol>
2. Hematoxylin-eosin (HE) staining and histopathology
<ol>
	<li>Four days after abdominal surgery, euthanize the mice with&#160;CO2 at a flow rate of 2 L/min in a CO2 chamber. Confirm death by cervical dislocation as a confirmatory method of euthanasia. Use surgical scissors to open the scrotum and remove the testes with forceps. Collect mouse testes 4 days after GSK923295 injection and fix them in 30 mL of 10% formaldehyde solution at room temperature for 12 h.</li>
	<li>For gradient dehydration, sequentially incubate the sample in 40 mL of 70% ethanol for 1 h, in 40 mL of 85% ethanol for 1 h, in 40 mL of 95% ethanol for 1 h, and in 40 mL of 100% ethanol for 1 h.</li>
	<li>Incubate the sample in 40 mL of xylene for 40 min, and then in 40 mL of paraffin for 1 h at 65 &#176;C. Place the tissues at the bottom of the embedding box. Add the melted paraffin into the embedding box. Cool the tissues for complete solidification at 4 &#176;C for 6 h.</li>
	<li>Fix the samples on the holder of the ultramicrotome, keep the angle between the samples and the knife surface at 5-10&#176;, and adjust the slice thickness to 5 &#181;m. Prepare the 5 &#181;m thick sections using an ultramicrotome, spread the slides in a water bath at 40 &#176;C, and dry the sections in a slide drier for 12 h at 37 &#176;C.</li>
	<li>Incubate the slides in 200 mL of xylene for 40 min, in 200 mL of 100% ethanol for 6 min, in 200 mL of 95% ethanol for 2 min, in 200 mL of 90% ethanol for 2 min, in 200 mL of 80% ethanol for 2 min, and in 200 mL of 70% ethanol for 2 min, respectively.</li>
	<li>Rinse the slides in distilled water for 5 min and stain them with the Mayer&#39;s hematoxylin solution for 6 min at room temperature. 
	NOTE: Mayer&#39;s hematoxylin solution: 0.011 mol/L hematoxylin, 6.7% anhydrous ethanol, 0.646 mol/L aluminum potassium sulphate, and 0.003 mol/L sodium iodate.</li>
	<li>Rinse the slides in running water for 5 min and incubate with distilled water for 2 min.</li>
	<li>Incubate the slides in 1% ethanol hydrochloride for 3 s, and then rinse them in running water for 2 min.</li>
	<li>Stain the sample with 1% eosin for 15 s, and then incubate them with 95% ethanol for 5 s, with 100% ethanol for 2 min, and in xylene for 40 min.</li>
	<li>Seal the slides using 15 &#181;L of neutral gum and the 24 x 50 mm coverslip.</li>
</ol>
3. Immunofluorescence and confocal microscopy
<ol>
	<li>Collect the 5 &#181;m thick paraffin sections of mouse testes for immunofluorescence. Incubate the slides in xylene for 40 min, in 100% ethanol for 6 min, in 95% ethanol for 2 min, in 90% ethanol for 2 min, in 80% ethanol for 2 min, and in 70% ethanol for 2 min. Rinse the slides in distilled water for 5 min, and rinse the slides with 0.01 M PBS for 5 min.</li>
	<li>Place the slides in the antigen retrieval solution (0.01 M citrate buffer) and boil under high pressure using a pressure cooker for 4 min for antigen retrieval. Cool the slides naturally to room temperature. Rinse with distilled water for 5 min twice, and with PBS for 5 min. 
	&#8203;NOTE: 0.01 M citrate buffer (pH 6.0): 2.1 mmol/L citric acid, 11.6 mmol/L trisodium citrate dihydrate.</li>
	<li>Permeabilize cells by incubating the slides in 500 &#181;L of 0.25% TritonX-100/PBS for 10 min. Rinse the slides with PBS for 5 min three times.</li>
	<li>For antigen blocking, incubate the samples with 300 &#181;L of 3% bovine serum albumin (BSA)/PBST (0.1% Tween-20 in PBS) for 1 h. Incubate the samples with the primary antibodies in 3% BSA/PBST for 16 h at 4 &#176;C. Put the slides in a humidified box to prevent the tissues from drying out. Rewarm the slides naturally to room temperature for 30 min.</li>
	<li>Discard the primary antibody, and then rinse the slides in PBST for 5 min three times. Dilute secondary antibodies in 3% BSA/PBST. Incubate the samples with secondary antibodies for 1-2 h at 37 &#176;C. Rinse the samples in PBST for 5 min five times.</li>
	<li>Stain the nuclei with 50 &#181;L of 4&#39;, 6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Mount the coverslip with the anti-fade mounting medium, and seal the coverslip with nail polish.</li>
	<li>Observe and record fluorescent signals in the slides using a fluorescent microscope equipped with a NA 40x/ 0.75 objective.</li>
</ol>
4. Flow cytometry
<ol>
	<li>Collect mouse testes in 6 cm Petri dishes and cut the testes into 1 mm3 pieces using surgical scissors.</li>
	<li>Digest the testes using 1 mL of 1% collagenase in 1.5 mL centrifuge tube for 10 min at 37 &#176;C, and then centrifuge the samples at 1,000 x g for 5 min to precipitate spermatogenic cells.</li>
	<li>Discard the supernatant, and then add 1 mL of 0.25% trypsin-EDTA (Ethylene Diamine Tetraacetic Acid) solution for 20 min at 37 &#176;C, and then centrifuge the samples at 1,000 x g for 5 min.</li>
	<li>Discard the supernatant, and then incubate the precipitated cells with 1 mL of 70% cold ethanol for more than 8 h at 4 &#176;C.</li>
	<li>Centrifuge the samples at 1,000 x g for 5 min, and then collect cell sediments. Stain the spermatogenic cells with 500 &#181;L propidium iodide (PI) staining solution (50 &#181;g/mL PI, 100 &#181;g/mL RNase A and 0.2% Triton X-100 in PBS) at 37 &#176;C for 30 min. 
	NOTE: Gently shake the centrifuge tube every 5 min to avoid cell aggregations.</li>
	<li>Filter the samples using a 300 mesh screen to get rid of cell debris; collect the cells in a flow tube and store them at 4 &#176;C.</li>
	<li>Detect fluorescence signals and light scattering at the excitation wavelength of 488 nm using a flow cytometer. Analyze the DNA content and light scattering using the Modfit MFLT32 software.</li>
</ol>
5. Transmission electron microscopy
<ol>
	<li>Cut the testes into 1 mm3 pieces using the sharp scalpel, and quickly incubate the samples with 3% glutaraldehyde-1.5% paraformaldehyde solution in 0.1 M PBS (pH 7.2) for 4 h at 4 &#176;C to avoid the changes of ultrastructure. Rinse the samples with 0.1 M PBS for 5 min three times. 
	NOTE: The scalpel and scissors should be sharp, and try to avoid artificial squeezing and pulling. The processing of samples should be carried out in the fixation fluid.</li>
	<li>Fix the samples in 1% osmic acid-1.5% potassium ferrocyanide solution at 4 &#176;C for 1.5 h. Dry the water with filter paper and rinse the samples with 0.1 M PBS for 5 min three times.</li>
	<li>Dehydrate the samples in 40 mL of 50% ethanol for 10 min at 4 &#176;C. Incubate the samples in 40 mL of 70% ethanol saturated uranium acetate dye at 4 &#176;C for 12 h, in 40 mL of 90% ethanol for 10 min at 4 &#176;C, in 40 mL of 90% ethanol-acetone for 10 min at room temperature, and in 40 mL of anhydrous acetone for 10 min three times at room temperature.</li>
	<li>Incubate the samples in anhydrous acetone-epoxy resin 618 embedding agents (v / v = 1:1) mixture for 1.5 h, and then embedded the samples in epoxy resin 618 embedding agents at 35 &#176;C for 3 h.</li>
	<li>For resin polymerization, incubate the samples in epoxy resin 618 embedding agents at 35 &#176;C for 12 h, at 45 &#176;C for 12 h, and then at 60 &#176;C for 24 h.</li>
	<li>Install the samples and the glass knife, and then adjust the distances between the samples and the knife. Prepare the 90 nm thick ultra-thin sections using an ultra-thin microtome. Slice the samples at a constant speed, and then place the slides on a nickel mesh. Place the slides in a Petri dish at room temperature.</li>
	<li>Stain the slides with 2% uranyl acetate for 10 min, and then stain the samples with 2% lead citrate for 10 min. Rinse the slides with distilled water. Dry the slides for 24 h at room temperature.</li>
	<li>Observe the slides and record electron images at 70-100 kV using a transmission electron microscope.</li>
</ol>

The authors have nothing to disclose.

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">
	<tbody>
		<tr>
			<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10009164</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Aluminium potassium sulphate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10001060</td>
			<td></td>
		</tr>
		<tr>
			<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>
		<tr>
			<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>
			<td></td>
		</tr>
		<tr>
			<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 ...

functional-assessment-kinesin-7-cenp-e-spermatocytes-using-vivo

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1.7K Views.  Fujian Medical University. 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.

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

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Centromere-Associated Protein-E Gene Editing Using the CRISPR/Cas9 System

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Author Spotlight: Establishing CENP-E Knockout HeLa Cells &#8211; A Novel Approach to Study Kinesin-7 CENP-E Biology and its Inhibitors

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...

Quantitative Analysis of Mitophagy in Murine Pancreatic &#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &#946;-Cells Using Effective and Robust Complementary Approaches 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...