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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

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Protokół

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

  1. Sterilize the surgical instruments at 121 °C for 30 min. Irradiate the surgical ultra-clean workbench with ultraviolet-C (UVC) 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.
  2. 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 during the surgery.
  3. 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. Secure the surgical area with a sterile drape.
  4. Disinfect the ventral abdomen with a Betadine scrub followed by 75% ethanol three times. Open the abdominal cavity using a sterile scalpel and make a <5 mm opening.
  5. 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 µL of GSK923295 into seminiferous tubules at a final concentration of 10 µM using 10 µL of rheodyne30. For the construction of the control group, inject 10 µL of 1% DMSO (dimethyl sulfoxide)/PBS (phosphate buffered saline) solution.
    NOTE: Store the GSK923295 solution at a concentration of 10 mM at -80 °C. Dissolve 0.1 µL of 10 mM GSK923295 into 100 µL of PBS solution to prepare the 10 µM of GSK923295 solution.
  6. 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.
  7. 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.
  8. 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.

2. Hematoxylin-eosin (HE) staining and histopathology

  1. Four days after abdominal surgery, euthanize the mice with 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.
  2. 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.
  3. Incubate the sample in 40 mL of xylene for 40 min, and then in 40 mL of paraffin for 1 h at 65 °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 °C for 6 h.
  4. Fix the samples on the holder of the ultramicrotome, keep the angle between the samples and the knife surface at 5-10°, and adjust the slice thickness to 5 µm. Prepare the 5 µm thick sections using an ultramicrotome, spread the slides in a water bath at 40 °C, and dry the sections in a slide drier for 12 h at 37 °C.
  5. 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.
  6. Rinse the slides in distilled water for 5 min and stain them with the Mayer's hematoxylin solution for 6 min at room temperature.
    NOTE: Mayer'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.
  7. Rinse the slides in running water for 5 min and incubate with distilled water for 2 min.
  8. Incubate the slides in 1% ethanol hydrochloride for 3 s, and then rinse them in running water for 2 min.
  9. 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.
  10. Seal the slides using 15 µL of neutral gum and the 24 x 50 mm coverslip.

3. Immunofluorescence and confocal microscopy

  1. Collect the 5 µm 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.
  2. 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.
    ​NOTE: 0.01 M citrate buffer (pH 6.0): 2.1 mmol/L citric acid, 11.6 mmol/L trisodium citrate dihydrate.
  3. Permeabilize cells by incubating the slides in 500 µL of 0.25% TritonX-100/PBS for 10 min. Rinse the slides with PBS for 5 min three times.
  4. For antigen blocking, incubate the samples with 300 µL 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 °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.
  5. 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 °C. Rinse the samples in PBST for 5 min five times.
  6. Stain the nuclei with 50 µL of 4', 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.
  7. Observe and record fluorescent signals in the slides using a fluorescent microscope equipped with a NA 40x/ 0.75 objective.

4. Flow cytometry

  1. Collect mouse testes in 6 cm Petri dishes and cut the testes into 1 mm3 pieces using surgical scissors.
  2. Digest the testes using 1 mL of 1% collagenase in 1.5 mL centrifuge tube for 10 min at 37 °C, and then centrifuge the samples at 1,000 x g for 5 min to precipitate spermatogenic cells.
  3. Discard the supernatant, and then add 1 mL of 0.25% trypsin-EDTA (Ethylene Diamine Tetraacetic Acid) solution for 20 min at 37 °C, and then centrifuge the samples at 1,000 x g for 5 min.
  4. Discard the supernatant, and then incubate the precipitated cells with 1 mL of 70% cold ethanol for more than 8 h at 4 °C.
  5. Centrifuge the samples at 1,000 x g for 5 min, and then collect cell sediments. Stain the spermatogenic cells with 500 µL propidium iodide (PI) staining solution (50 µg/mL PI, 100 µg/mL RNase A and 0.2% Triton X-100 in PBS) at 37 °C for 30 min.
    NOTE: Gently shake the centrifuge tube every 5 min to avoid cell aggregations.
  6. 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 °C.
  7. 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.

5. Transmission electron microscopy

  1. 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 °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.
  2. Fix the samples in 1% osmic acid-1.5% potassium ferrocyanide solution at 4 °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.
  3. Dehydrate the samples in 40 mL of 50% ethanol for 10 min at 4 °C. Incubate the samples in 40 mL of 70% ethanol saturated uranium acetate dye at 4 °C for 12 h, in 40 mL of 90% ethanol for 10 min at 4 °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.
  4. 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 °C for 3 h.
  5. For resin polymerization, incubate the samples in epoxy resin 618 embedding agents at 35 °C for 12 h, at 45 °C for 12 h, and then at 60 °C for 24 h.
  6. 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.
  7. 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.
  8. Observe the slides and record electron images at 70-100 kV using a transmission electron microscope.

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Wyniki

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...

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Dyskusje

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...

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

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).

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Materiały

NameCompanyCatalog NumberComments
0.25% Trypsin-EDTAGibco25200056
1 ml SyringeSeveral commercial brands availableSterile.
1.5 mL Centrifuge tubeAxygenMCT-150-C
50 mL Centrifuge TubeCorning430828
6 cm Petri dishCorning430166
95% EthanolSinopharm Chemical Reagent Co.,Ltd10009164
tubulin rabbit polyclonal antibodyBeyotimeAF0001For immunofluorescence assays. Use at 1:100.
rabbit anti-Histone H3 (phospho S10) monoclonal antibodyAbcamab267372For immunofluorescence assays. Use at 1:100.
rabbit anti-TUBA4A polyclonal antibodySangon BiotechD110022For immunofluorescence assays. Use at 1:100.
Anti-SYCP3 rabbit monoclonal antibodyAbcamab175191For immunofluorescence assays. Use at 1:100.
Adhesion microscope slidesCITOTEST188105
Alexa fluor 488-labeled goat anti-rabbit antibodyBeyotimeA0423Sencodary antibody. Use at 1:500.
Aluminium potassium sulphateSinopharm Chemical Reagent Co.,Ltd10001060
Anhydrous ethanolSinopharm Chemical Reagent Co.,Ltd100092690
Anti-fade mounting mediumBeyotimeP0131Prevent photobleching of flourescent signals.
BD FACS Canto IIBD BiosciencesFACS Canto II
Bovine Serum AlbuminSinopharm Chemical Reagent Co.,Ltd69003435
CentrifugeEppendorf5424BK745380
Chloral hydrateSinopharm Chemical Reagent Co.,Ltd80037516
Citric acidShanghai Experiment Reagent Co., Ltd122670
CollagenaseSangon BiotechA004194-0100
CoverslipsCITOTEST10212020C20 × 20 mm. Thickness 0.13-0.16 mm.
DAPIBeyotimeC1006
Dye vatSeveral commercial brands available91347802
Eosin Y, alcohol solubleSinopharm Chemical Reagent Co.,Ltd71014460
EtherSinopharm Chemical Reagent Co.,Ltd10009318
Formaldehyde - aqueous solutionSinopharm Chemical Reagent Co.,Ltd10010018
GSK923295MedChemExpressHY-10299
Hematoxylin, anhydrousSinopharm Chemical Reagent Co.,Ltd71020784
ICR mouseShanghai SLAC Laboratory Animal Co., Ltd
Image J softwareNational Institutes of Healthhttps://imagej.nih.gov/ij/Fluorescent image analysis.
Leica ultramicrotomeLeica
Leica EM UC-7 ultramicrotomeLeicaEM UC7
Modfit MFLT32Verity Software HouseFor analysis of flow cytometry results.
Nail polishSeveral commercial brands available
Neutral gumSinopharm Chemical Reagent Co.,Ltd10004160
Nikon Ti-S2 microscopeNikonTi-S2
Picric acidSinopharm Chemical Reagent Co.,LtdJ60807
RheodyneSangon BiotechF519160-000110 μl rheodyne
Sliced paraffinSinopharm Chemical Reagent Co.,Ltd69019461
Sodium iodateSinopharm Chemical Reagent Co.,Ltd80117214
Surgical instrumentsSeveral commercial brands availableFor abdominal surgery. Sterilize at 121 °C, 20 min.
Transmission electron microscopeFEITecnai G2
Trisodium citrate dihydrateShanghai Experiment Reagent Co., Ltd173970
Triton X-100Sinopharm Chemical Reagent Co.,Ltd30188928Dilute in sterile PBS to make a 0.25% working solution.
Tween 20Sinopharm Chemical Reagent Co.,Ltd30189328Dilute in sterile PBS to make a 0.1% working solution.
ParaformaldehydeSinopharm Chemical Reagent Co.,Ltd80096618
XyleneSinopharm Chemical Reagent Co.,Ltd10023418

Odniesienia

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