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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 se....

Protocol

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 12.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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 n....

Materials

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

References

  1. Lenormand, T., Engelstädter, J., Johnston, S. E., Wijnker, E., Haag, C. R. Evolutionary mysteries in meiosis. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 371 (1706), 20160001 (2016).
  2. Brandeis, M.

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