Name: functional assessment of kinesin-7 cenp-e in spermatocytes using in vivo inhibition, immunofluorescence and flow cytometry
Uploaded: 2021-12-28T14:00:00.000+00:00
Duration: 9 min 41 s
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;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 operate and highly reproducible. In addition, the integrity of testes are maintained, which is suitable for the studies of intact tissues in the context of the organs.
There are several critical steps in this protocol. For example, it is important to maintain a sterile environment during abdominal surgery for the prevention of postoperative infections31. Furthermore, for mice of different ages or different experimental animals, the amounts of injection should be adjusted appropriately according to the size of the testes and the effectiveness of the drugs19,32. In addition, the proper digestion time and microscopic observation is required for the determination of single cells and subsequent cell population testing during flow cytometry. However, there are several limitations in this protocols. It is difficult to use abdominal surgery to construct a mouse model when the age of the mouse is less than 2 weeks old. The main reasons are that the mice are too young, the postoperative diet is difficult and the survival rate is low. The drugs used in animal models are liquid and have the characteristics of easily penetrating cell membranes19,32,33, which are suitable for the applications of this administration method.
Understanding meiosis is stimulated by in vitro cell culture and gene knockout studies, however, it is not easily applicable in mammalian spermatocytes28. The obstacle to the mechanistic studies of male meiosis has been the lack of an appropriate system for manipulation and observation of dividing spermatocytes in meiosis27. The mouse is an excellent model organism in the research of cellular and molecular mechanisms of meiosis during spermatogenesis. The first wave of mouse spermatocytes starts meiosis at day 10 post-partum (dpp) and develops into mature sperms at 35 dpp, which provides a developmental time-window for the studies of meiotic division and spermatogenesis34.
Testis injection, including direct injection through the scrotum and microinjection via the abdominal surgery, is a useful technique for the studies of spermatogenesis35,36. Direct injection through the scrotum is quick and simple, and only causes a minor surgical trauma, which is suitable for mice of 4-week-old or older with the testes descend to the scrotum. However, it is impossible to inject the undescended testis of the 3-week-old or younger mice. Microinjection through the intraperitoneal surgery or scrotum surgery is suitable for the injection of undescended testes, which requires proficient surgical skills of experimental operators, a stereomicroscope, and the surgical and laboratory equipment. According to the injection sites, microinjection can be classified into seminiferous tubule injection, testicular net injection and testicular interstitial injection. Compared with other injection-based testis models, the injection of inhibitors through abdominal surgery can effectively inhibit the functions of proteins and have the advantages in cell membrane penetration, easy procedures, and long-term inhibition19,32. The methods using siRNAs, antisense oligonucleotides or lentivirus have greater limitations in low penetration efficiency of cell membranes, off-target side effects, and easy degradation of siRNA or oligonucleotides in vivo37,38,39,40.
The meiotic division in living tissues is more complex than that in culture conditions, in which the tissue architecture, the developmental signaling and environmental factors in vivo should be taken into consideration41. Previous studies have demonstrated that culture media and cell autonomous factors are crucial for the stimulation of the onset of meiotic division phase. For example, the phosphatase inhibitor okadaic acid (OA)42, spermatocyte-specific histone HIST1H1T43, topoisomerase II44, and metaphase promoting factor (MPF)45 promote the G2/MI transition in cultured spermatocytes. These complex culture conditions and factors contribute to the limitations of short-term culture of spermatocytes.
Direct injection of plasmid DNA into testes is successfully applied in testis mediated gene transfer and the production of transgenic mice32,46. The in vivo electroporation involves the injection of a DNA expression plasmid into the lumen of seminiferous tubules, and then use a series of electric pulses to change cell membrane permeability, improve the efficiency of transgene expression, and genetic modification45,46,47,48,49. Our method could be combined with in vivo electroporation, as well as fluorescent tagged proteins and gene editing tools, making this approach more powerful for the analyses of male meiotic division in the physiological context of tissues and organs.

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><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 &amp;times; 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 &amp;mu;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 &amp;deg;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, in the GSK923295 group, the spermatogenic wave were altered in the seminiferous tubules, and the metaphase arrested primary spermatocytes were significantly increased after CENP-E inhibition (Figure 2B-D). Importantly, several homologous chromosomes were not aligned at the equatorial plate after CENP-E inhibition (Figure 2B-D). Furthermore, CENP-E inhibition also led to the increase of metaphase I spermatocytes in seminiferous tubules (Figure 2E,F,G). Taken together, CENP-E inhibition results in chromosome misalignment in primary spermatocytes during meiosis I, which suggests that CENP-E is responsible for chromosome congression and alignment of spermatocytes in meiosis.
To further validate these results, we performed the immunofluorescence assays to detect the spermatogenic cells in seminiferous tubules. We found that the regular spermatogenic wave shown in the control group was obviously changed and became irregular in the GSK923295 group (Figure 3). The meiotic spindle of dividing spermatocytes was labeled with anti-&#945;-tubulin antibody, and the transverse filament of synaptonemal complexes in the spermatocytes was labeled with anti-synaptonemal complex protein 3 (SYCP3) antibody (Figure 3A). The SYCP3 positive cells per seminiferous tubule decreased after CENP-E inhibition (Figure 3B). Meanwhile, the SYCP3 dots per metaphase cell were not disrupted after CENP-E inhibition (Figure 3C). In addition, the SYCP3 stretches per cell were also not affected in the GSK92395 groups (Figure 3D). Strikingly, we found that the distances of spindle poles in metaphase I spermatocytes were increased after CENP-E inhibition (Figure 3E,F). These immunofluorescent results suggest that CENP-E is required for chromosome misalignment and the processes of spermatogenesis, and is indispensable for the maintenance of bipolar spindle and the organization of the meiotic spindle.
To investigate cell populations in mouse testes, we digested the testes and performed PI staining and flow cytometry assays (Figure 4). The important technical procedures were shown in Figure 4A-C. The spermatogenic cells consist of several cell populations, including the spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa. The DNA contents of these spermatogenic cells were shown in Figure 4A. We demonstrated that CENP-E inhibition resulted in the decrease of the haploid cells from 42.95 &#177; 1.09% in the control group to 38.26 &#177; 1.86% in the GSK923295 group (Figure 4B-D). The ratios of the diploid cells and aneuploidy cells were not significantly influenced after CENP-E inhibition (Figure 4E,F). Furthermore, the ratios of the tetraploid cells increase from 17.76 &#177; 1.52% in the control group to 28.88 &#177; 2.05% in the GSK923295 group (Figure 4G). Taken together, we find that cell populations of the spermatogenic cells are slightly altered after CENP-E inhibition. CENP-E inhibition results in the decrease of the haploid cells and the increase of tetraploid cells, which indicates that CENP-E inhibition is associated with metaphase arrest in the dividing spermatocytes.
Furthermore, we observed the submicroscopic structure of the spermatogenic cells using transmission electron microscopy (Figure 5). The chromatin organization, endoplasmic reticulum and mitochondria of spermatocytes were shown in Figure 5. We found that the organization of spermatogenic cells was slightly disrupted in the GSK923295 group (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63271/63271fig01.jpg" /> 
Figure 1: Establishment of an in vivo inhibition model of mouse testes through abdominal surgery and testicular administration. (A) All surgical instruments used in abdominal surgery. 1) dissecting scissor, 2) needle forceps, 3) straight forceps, 4) pincett, 5) rheodyne, 6) 1 mL syringe, 7) Scalpel with no. 3 handle and no. 11 blade, 8) stylolite, 9) round stitching needles, 1/2 0.6 x 14 mm, 1/2 0.7 x 17 mm, 10) ethanol swabs. (B) After anesthesia, the mice were placed in a supine position on a wax tray, the lower abdomen was prepared and disinfected with 75% ethanol. (C) A &#60;5 mm opening was made in the middle of the lower abdomen. (D) The epididymal fat pad was pulled with sterile dissecting forceps to locate the testes. The testis was injected with 10 &#181;L GSK923295 using a 10 &#181;L rheodyne. (E) The peritoneum and skin was simultaneously sutured with two stitches. (F) The wound was disinfected with 75% ethanol. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63271/63271fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63271/63271fig02.jpg" /> 
Figure 2: CENP-E inhibition resulted in the arrest of meiosis I and chromosome misalignment in mouse spermatocytes. (A) HE staining of the spermatogenic cells in the control group. The arrows indicate the spermatocytes. (B) HE staining of the spermatogenic cells in the GSK923295 group. The testes were injected with GSK923295 for 4 days at a final concentration of 10 &#181;M. The arrows indicate chromosome misalignment in the spermatocytes. For all images, scale bar, 10 &#181;m. (C) Ratio of metaphase spermatocytes in seminiferous tubules. Control, 13.43 &#177; 1.68%; GSK923295, 42.29 &#177; 3.94%. N = 1308 cells were analyzed. Group = 4. Student&#39;s t-test. Error bars, means &#177; SEM. ***, P &#60; 0.001. (D) Ratio of seminiferous tubules with dividing spermatocytes. Control, 5.38 &#177; 2.64%; GSK923295, 33.96 &#177; 3.87%. N = 151 seminiferous tubules were analyzed. Group = 4. (E) Immunofluorescence images of Histone H3 (phospho Ser 10) and TUBA4A in the control and GSK923295 groups. TUBA4A, red; Histone H3 (phospho Ser 10), green; DAPI, blue. Scale bar, 10 &#181;m. The enlarged box is zoomed. (F) The quantification of the number of metaphase cells in seminiferous tubules. Control, 11.45 &#177; 1.55; GSK923295, 18.91 &#177; 2.36. N = 11. (G,H) Line-scan analyses of fluorescent intensities of Histone H3 and TUBA4A in metaphase I spermatocytes of the control group (G) and the GSK923295 group (H). TUBA4A, red; Histone H3 (phospho Ser 10), green; DAPI, blue. The X axis indicates the relative distance. The Y axis indicates fluorescent intensities. Student&#39;s t-test. Error bars, mean &#177; SEM. *, P &#60; 0.05; ***, P &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63271/63271fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63271/63271fig03.jpg" /> 
Figure 3: CENP-E inhibition by GSK923295 led to the disorganization of seminiferous tubules and the disruption of spermatogenesis. (A) Representative immunofluorescence images of SYCP3 and TUBA4A in the control and GSK923295 groups. SYCP3, red; TUBA4A, green; DAPI, blue. Scale bar, 10 &#181;m. (B) SYCP3 positive cells per seminiferous tubules in the control and GSK923295 groups. Control, 56.00 &#177; 5.43%; GSK923295, 39.00 &#177; 1.73%. N = 10. (C) The quantifications of SYCP3 dots per metaphase cells. Control, 10.00 &#177; 1.02; GSK923295, 9.71 &#177; 0.86, N = 7. (D) The quantifications of SYCP3 stretches per cell in the control and GSK923295 groups. Control, 8.63 &#177; 0.22; GSK923295, 8.21 &#177; 0.21. N = 19. (E) The analyses of the distance of spindle poles in the metaphase I spermatocytes. Control, 8.20 &#177; 0.28 &#181;m; GSK923295, 9.30 &#177; 0.29 &#181;m. N = 30. (F) Immunofluorescence images of seminiferous tubules in the control and GSK923295 groups. The arrows indicate the spermatocytes. DAPI, green; &#946;-tubulin, green. The enlarged images of the dashed box were shown in the zoom. For all images, scale bar, 10 &#181;m. Student&#39;s t-test. Error bars, mean &#177; SEM. ns, P &#62; 0.05; **, P &#60; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63271/63271fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63271/63271fig04.jpg" /> 
Figure 4: Flow cytometry analysis of spermatogenic cells in mouse testes. (A) Schematic illustrations of the key steps in cell cycle analysis of mouse spermatogenic cells. The diploid spermatocytes undergo meiosis I and II to form the haploid spermatids. The C value indicates the DNA content. The N value (ploidy) indicates the number of sets of chromosomes. (B,C) Flow cytometry analysis of the spermatogenic cells in the control group (B) and the GSK923295 group (C). For in vivo CENP-E inhibition, GSK923295 was injected into 3-week-old male ICR mouse testes at a final concentration at 10 &#181;M for 4 days. For cell cycle analysis, n = 3,000 cells were measured and analyzed. P4, the haploid cells (1C). P5, the diploid cells (2C). P6, the tetraploid cells (4C). (D) Ratios of the haploid cells in the control and GSK923295 groups. Control, 42.95 &#177; 1.09%; GSK923295, 38.26 &#177; 1.86%. N = 8. (E) Ratios of the diploid cells in the control and GSK923295 groups. Control, 20.10 &#177; 0.91%; GSK923295, 17.95 &#177; 0.81%. N = 8. (F) Ratios of the aneuploid cells (2C ~ 4C) in the control and GSK923295 groups. Control, 3.41 &#177; 0.23%; GSK923295, 3.39 &#177; 0.25%. N = 8. (G) Ratios of the tetraploid cells in the control and GSK923295 groups. Control, 17.76 &#177; 1.52%; GSK923295, 28.88 &#177; 2.05%. N = 8. For all graphs, Student&#39;s t test. Error bars, mean &#177; SEM. ns, P &#62; 0.05; *, P &#60; 0.05; ***, P &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63271/63271fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63271/63271fig05.jpg" /> 
Figure 5: Electron microscopy analysis of the spermatogenic cells in the control and GSK923295 group. (A) Representative images of seminiferous tubules in the control group. Scale bar, 5 &#181;m. (B) The enlarged image of the nucleus of the spermatocyte. The arrows indicate homologous chromatin of the spermatocytes. Scale bar, 1 &#181;m. (C) The enlarged image of the cytoplasm of the spermatocytes. Scale bar, 1 &#181;m. (D) Representative images of seminiferous tubules in the GSK923295 group. The testes were treated with 10 &#181;M GSK923295 for 4 days. Scale bar, 5 &#181;m. (E) The enlarged image of the nucleus of the spermatocyte in the GSK923295 group. Scale bar, 1 &#181;m. (F) The enlarged image of the cytoplasm of the spermatocytes in the GSK923295 group. Scale bar, 1 &#181;m. For all graphs, sc, spermatocyte; sd, spermatid. ER, endoplasmic reticulum; mt, mitochondria. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63271/63271fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

1.7K Views.  Fujian Medical University. Meiose is een evolutionair geconserveerde gebeurtenis bij eukaryoten, die essentieel is voor gametogenese en seksuele voortplanting. Ons protocol biedt een effectieve methode voor studies van meiotische deling en spermatogenese. We hebben een reeks protocollen beschreven voor de analyse van spermatocyten.Deze techniek kan worden toegepast voor observatie van meiotische spoel, homologe chromosomen en de subcellulaire organellen in spermatocyten. Deze methode kan worden gebruikt voor de ...

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

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Biochemistry

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

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.

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 ...

Developmental Biology

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

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

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