Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Summary

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Abstract

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Introduction

Proper segregation of chromosomes during mitosis and meiosis requires that sister chromatid cohesion be established, maintained, and released in a coordinated fashion^1,2. Cohesion is established during S phase and is mediated by the cohesin complex, which forms physical linkages that hold the sister chromatids together. In meiosis, cohesion distal to a crossover also functions to hold recombinant homologs together and this physical association helps ensure proper orientation of the bivalent on the metaphase I spindle (Figure 1)^3,

Protocol

1. Preparations

1. Prepare solutions for fluorescence in situ hybridization (FISH). Prepare all solutions using ultrapure water.
   1. Prepare 5x Modified Robb's buffer: 275 mM potassium acetate, 200 mM sodium acetate, 500 mM sucrose, 50 mM glucose, 6 mM magnesium chloride, 5 mM calcium chloride, 500 mM HEPES pH = 7.4. Bring the pH to 7.4 using 10 N NaOH. Filter sterilize and store at -20 °C. Thaw and dilute to 1x as needed and store 1x aliquots at -20 °C.
   2. Prepare 10x Phosphate buffered saline (PBS) using 1.3 M NaCl, 70 mM Na₂HPO₄, 30 mM NaH₂PO_4<....

Results

Figure 5 presents images obtained with an arm probe that hybridizes to cytological region 6E-7B on the X chromosome. This probe results in a signal that co-localizes with that of DAPI, is easily distinguishable from the background, and has been used successfully to quantify arm cohesion defects in different genotypes¹⁹. Quantification of cohesion defects was restricted to prometaphase I and metaphase I stages; oocytes prior to.......

Discussion

The use of FISH probes to assess the state of arm cohesion in prometaphase I and metaphase I Drosophila oocytes is a significant advancement in the field of Drosophila meiosis. Historically, Drosophila researchers have been limited to genetic tests to infer premature loss of arm cohesion in mature oocytes^11,18,19. Now, with the methods presented here, the state of arm cohesion can be assayed directly u.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Kits
Midi Prep kit	Qiagen	12143	Prep BAC clone DNA
GenomePlex Complete Whole Genome Amplification (WGA) Kit	Sigma	WGA2	Amplify BAC clone DNA
ARES Alexa Fluor 647 DNA labeling kit	Invitrogen	A21676	Label BAC clone DNA
PCR purification kit	Qiagen	28104	Remove non-conjugated dye following labeling of BAC clone DNA
Name	Company	Catalog Number	Comments
Chemicals & Solutions
Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.
Bovine serum albumin (BSA)	Fisher Scientific	BP1600-100	Prepare 10% stock Freeze aliquots
Calcium chloride	Fisher Scientific	C75-500
DAPI (4’, 6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen	D1306	Toxic: wear appropriate protection. Prepare 100µg/ml stock in 100% ethanol, store in aliquots at -20 °C. Prepare 1 µg/ml solution in 2X SSCT before use.
Dextran sulfate	Sigma	D-8906
Drierite	Drierite Company	23001
Dithiothreitol (DTT)	Invitrogen	15508-013	Prepare 10 mM stock
dTTP (10 µmol, 100 µl)	Boehringer Mannheim	1277049	Prepare 1 mM stock
EDTA (Disodium ethylenediamine tetraacetic acid)	Fisher Scientific	S311-500	Prepare 250 mM stock
100% ethanol (molecular grade, 200 proof)	Decon Laboratories	2716
Tris (Ultra Pure)	Invitrogen	15504-020
EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid)	Sigma	E-3889
16% formaldehyde	Ted Pella, Inc.	18505	Toxic: wear appropriate protection
Formamide	Invitrogen	AM9342	Toxic: wear appropriate protection
Glucose	Fisher Scientific	D16-1
Glycogen	Roche	901393
HEPES	Boehringer Mannheim	737-151
Heptane	Fisher Scientific	H350-4	Toxic: wear appropriate protection.
Hydrochloric acid	Millipore	HX0603-4	Toxic: wear appropriate protection.
Hydroxylamine	Sigma	438227	Prepare 3 M stock
4.9 M Magnesium chloride	Sigma	104-20
Na2HPO4 Ÿ 7H2O	Fisher Scientific	S373-500
NaH2PO4 Ÿ 2H2O	Fisher Scientific	S369-500
Poly-L-lysine (0.1mg/ml)	Sigma	P8920-100
Potassium acetate	Fisher Scientific	BP364-500
Sodium acetate	Fisher Scientific	S209-500	Prepare 3M stock
Sodium cacodylate	Polysciences, Inc.	1131	Toxic: wear appropriate protection. Prepare 400mM stock
Sodium citrate	Fisher Scientific	BP327-1
Sodium chloride	Fisher Scientific	S271-3	Sodium chloride
Sucrose	Fisher Scientific	S5-500
10% Tween 20	Thermo Scientific	28320	Surfact-Amps
10% Triton X-100	Thermo Scientific	28314	Surfact-Amps
Name	Company	Catalog Number	Comments
Solutions
Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.
TE buffer			10 mM Tris, 1 mM EDTA, pH = 8.0
20X SSC (Saline Sodium Citrate)			3 M NaCl, 300 mM sodium citrate
2X cacodylate fix solution			Toxic: wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA
1.1X Hybridization buffer			3.3X SSC, 55% formamide, 11% dextran sulfate
Fix solution			Toxic: wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution
PBSBTx			1X PBS, 0.5% BSA, 0.1% Trition X-100
PBSTx			1X PBS, 1% Trition X-100
Extraction buffer (PBSTx + Rnase)			1X PBS, 1% Trition X-100, 100 µg/mL RNase
2X SSCT			2X SSC, 0.1% Tween 20
2X SSCT + 20% formamide			Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide
2X SSCT + 40% formamide			Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide
2X SSCT + 50% formamide			Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide
Name	Company	Catalog Number	Comments
Enzymes
AluI	New England Biolabs	R0137S
HaeIII	New England Biolabs	R0108S
MseI	New England Biolabs	R0525S
MspI	New England Biolabs	R0106S
RsaI	New England Biolabs	R0167S
BfuCI	New England Biolabs	R0636S
100X BSA	New England Biolabs		Comes with NEB enzymes
10X NEB buffer #2	New England Biolabs		Restriction enzyme digestion buffer. Comes with NEB enzymes
Terminal deoxynucleotidyl transferase (TdT) 400 U/µl	Roche/Sigma	3333566001
TdT buffer	Roche/Sigma		Comes with TdT enzyme
Cobalt chloride	Roche/Sigma		Toxic: wear appropriate protection. Comes with TdT enzyme
RNase A (10 mg/mL)	Thermo-Scientific	EN0531
Name	Company	Catalog Number	Comments
Cytology Tools etc.
Forceps	Dumont		#5 INOX, Biologie
9” Disposable glass Pasteur pipettes	Fisher	13-678-20C	Autoclave to sterilize
Shallow glass dissecting dish			Custom made
Deep well dish (3 wells)	Pyrex	7223-34
Fisherfinest Premium microscope slides	Fisher Scientific	22-038-104	Used to cover deep well dishes
Frosted glass slides, 25 x 75 mm	VWR Scientific	48312-002
Glass slides, 3 x 1 in, 1 mm thick	Thermo-Scientific	3051
Coverslips, 10 x 10 mm, No. 1.5	Thermo-Scientific	3405
Tungsten needle			homemade
Prolong GOLD mounting media	Molecular Probes	P36930
Compressed-air in can	Various
Name	Company	Catalog Number	Comments
Equipment
PCR machine	Various
Nanodrop 2000, spectrophotometer	Thermo-Scientific		microvolume spectrophotometer
Vortexer	Various
Table top microfuge at room temperature	Various
Table top microfuge at 4 °C	Various
Heat block	Various
Hybridization oven or incubator with rotator	Various
Nutator	Various
A1RSi laser scanning confoal	Nikon		40X oil Plan Fluor DIC (NA 1.3)
Name	Company	Catalog Number	Comments
Consumables etc.
50 mL conical tubes	Various
15 mL conical tubes	Various
1.5 mL microfuge tubes	Various
500 µl microfuge tubes	Various
200 µl PCR tubes	Various
Plastic container with tight fitting lid	Various		To hold Drierite
Kimwipes	Various		disposable wipes
Parafilm	Various		paraffin film
Name	Company	Catalog Number	Comments
Other
HPLC purified 5'-labeled oligonucleotides	Integrated DNA Technologies		Cy3-labeled probes that recognize the 359 bp satellite repeat of the X chromosome
Volocity 3D Image Analysis Software	PerkinElmer		Version 6.3