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

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 diversity^1,2,3. 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 I^4,5. 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.......

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.......

Materials

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