Induction of chromosomal fragile sites in goats: a preliminary study

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Induction of chromosomal fragile sites in goats: a

preliminary study

Nl López-Corrales, Mv Arruga

To cite this version:

’

’’Original

article

Induction of chromosomal

_fragile

sites

in

_goats:

a

preliminary study

NL

_{López-Corrales,}

MV

_Arruga*

Departamento

de Producci6n Animal _y Ciencias de los

_Alimentos,

Facultad de Veterinaria de la Universidad de

_Zaragoza,

_c/

_Miguel

Servet

_177,

5001,i

_{Zaragoza, Spain}

(Received

9 November 1995;

accepted

16

_January

₁₉₉₆₎

Summary - The current

study

describes the results obtained from different methods of detection of folate-sensitive

_fragile

sites in goat chromosomes. Two different types of

expression

of chromosomal

_fragility

have been observed: telomeric

_non-staining

gaps, in 20

out of 21 animals

studied,

and chromatidic breaks in ten animals. The

non-staining

gaps have been identified

_mainly

in chromosome

5,

and their

_frequency

of occurrence

_ranged

from 30 to 66% of the cells. The chromatidic break occurrence

_ranged

from 2 to 5% of the cells _among the break carriers. From the methods used and the observed

_frequency

of

_expression

in

_cultures,

the gaps were classified as common folate-sensitive

_fragile

sites.

Significant

differences between the induction methods used were obtained.

’

goat

/

fragile site

_/

folate _deficiency

Résumé - Induction de sites chromosomiques fragiles chez les chèvres : étude

préliminaire. Cette étude décrit les résultats de

différentes

méthodes de mise en évidence de sites

_{chromosomiques fragiles}

sensibles au

folate

chez la chèvre. Deux _types

différents

d’expression

de la

_{fragilité chromosomique}

ont été observés : des espaces

télomériques

ne

prenant pas la

coloration,

sur 20 des 21 animaux

_étudiés,

et des cassures

_{chromatidiques}

sur 10 animaux. Les absences de coloration ont été localisées

_{principalement}

sur le chro-mosome 5 et leur

_{fréquence d’apparition}

allait de 30 à 66 % des cellules. Chez les por-teurs de cassures, la

fréquence

de ces dernières allait de 2 à 5 % des cellules.

_D’après

les

méthodes utilisées et les

_fréquences

observées dans les

cultures,

les zones

_{chromosomiques}

non colorées _peuvent être considérées comme appartenant à la

_catégorie

commune des sites

fragiles

sensibles

_{au folate.}

Des

_{différences significatives}

entre les méthodes d’induction ont

également

été observées.

chèvre

_/

site _fragile

_/

déficience en folate

’"

INTRODUCTION

About one hundred chromosomal

_fragile

sites have been detected in humans since

the first

_description

was made

_by

Dekaban in 1965

_(Sutherland,

_1991).

Human

fragile

sites have been

_successively

related to different

_pathologies

and one of

the most well known is the association between the mental retardation

_syndrome

and the

_fra.Xq27.3

_(Sutherland

and

_Baker,

1990;

Vogel

et

al, 1990;

Oberl6 et

al,

1991;

Craig,

1991).

Furthermore,

implications

of chromosomal

_fragility

in different

processes like Bloom

syndrome

(Fundia

et

_al,

_1992),

chromosomal viral

integration

points

(Caporossi

et

_al,

_1991),

chromosomal evolution

_(Mir6

et

_{al, 1987;}

_Popescu

et

al,

1990)

and the

_relationship

between

_{fragile sites, oncogenesis}

and tumoral events

(Yunis,

1983;

Yunis and

_{Soreng, 1984; DeBraeckeler, 1987;}

Dal Cin et

_{al, 1991;}

Austin et

_al,

₁₉₉₁₎

have been well documented in human

_{cytogenetics.}

In animals

only

a few chromosomal

fragile

sites have been

_{reported, mainly}

in domestic

_species.

In

_pigs,

_Riggs

and Chrisman

_{(1989, 1991)}

have described

_aphidicolin

and folate-sensitive

_fragile

sites like the ones detected

_by

Yan and

_Long

(1993).

More

recently,

folate,

5-BrdU and

_{aphidicoline fragiles}

sites have been found in

_{equine, rabbit,}

bovine,

mole

_rat,

_dog

and

_sheep

_{karyotypes by}

Ronne

_(1992),

Poulsen and

Ronne

(1991),

Uchida et al

_(1986),

Gripemberg

(1991),

Stone et al

_(1991a)

and

_Matejka

et

al

_(1990).

_Although

no close

_relationship

with _any

_pathology

has

_yet

been

_observed,

Tewari et al

₍₁₉₈₇₎

have indicated a

_possible

effect on the

_fertility

of female rats and

Stone et al

_(1991b)

have

_suggested

the

_implication

of some

fragile

sites in tumoral

chromosomal

_{rearrangements}

in the _mammary

_glands

of

_dogs.

These features and

some of the

published results,

indicate that the

fragile

sites _may be distributed in

the

_majority

of domestic

_species

in a similar _way as for humans. This

highlights

the

importance

of

_knowing

the distribution and

_{morphological}

characteristics of animal

fragile

sites,

as a first

_step

to

_finding

the

possible

relationships

between any defined

pathology

or

syndrome

and the _presence of chromosomal

fragility.

There is no

_knowledge

of the induction

methodologies

or chromosomal

_fragility

expression

forms in

_goats

and the aim of this work has been the

_adaptation

of

induction

methodologies

to

begin

studies of the detection and identification of folate-sensitive

_fragile

sites in this

_karyotype.

MATERIAL AND METHODS

Twenty-one

adult

goats

were

_{used, including}

_Saanen,

_Toggenburg

and cross-bred

animals.

An

_adaptation

of

_published

_protocols

_(Sutherland

et al

_(1985);

Howard-Peebles

(1991);

Fisch et al

_(1991);

_Jacky

et al

_(1991))

was used to induce the

_expression

of

fragile

sites in

_lymphocyte

cultures.

Whole blood

_{(1 mL)}

was cultivated in 10 mL of low folate M-199 medium

_(Flow)

supplemented

with

5%

SFB

_(Gibco),

1%

_{penicillin-streptomycin}

_(Gibco),

5 IU of

PHA

_{(phytohaemaglutinine) (Wellcome)}

and 5 IU of Pokeweed

_(Gibco)-like

mi-togens.

The culture

_pH

was

_adjusted

to 7.6-7.8

_by

the addition of bicarbonate. Three modifications to this basic culture were used:

protocol

1: 5

_vM

of

were added

_during

the last 24 h of cell

_{culture; protocol}

2: 5

_R

_M

of

fluorodeoxyuri-dine

_(Sigma,

F

_5030),

30

_mg/mL

of

_thymidine

_(Sigma,

T

₅₀₁₈₎

and 10

_!g/mL

5-bromo-2’-deoxyuridine

BrdU

_(Sigma,

B

₅₀₀₂₎

were added

during

the last 24 h of

cell

_{culture; protocol}

3: 5

_R

_M

of

_{fluorodeoxyuridine}

_(Sigma,

F

_5030),

30

_mg/mL

of

thymidine

(Sigma,

T

_5018),

and

10-

5

M

of

_amethopterin

and methotrexate

_(Sigma

A

₆₇₇₀₎

were added

_during

the last 24 h of cell culture.

The cultures were harvested and fixed

according

to a standard

technique

(Moor-head et

_al,

_1960).

Two cultures for each treatment were made and 50 cells from

each one were observed. Control cultures were used for each

_protocol

_according

to

the standard

_methodology:

1 mL of whole blood in 10 mL of RPMI 1640

_(Gibco),

supplemented

with

20%

SFB

_(bovine

calf

_serum)

_(Gibco)

and

1%

penicilline-streptomycin

(Gibco)

and

1%

_L

_-glutamine

_(Sigma).

The cultures were incubated

at 37 °C in the absence of

C0

2

and harvested after 72 h of

_growth.

The identification of chromosome

_pairs

was

accomplished by

an

adaptation

of

the

_{original Seabright’s}

G

_banding

method

_{(Seabright, 1971).}

An ANOVA test was

used to establish the differences between treatments

_(Stat

_View,

_Macintosh).

RESULTS

Two different

_types

of chromosomal alterations were observed. The numbers

_given

below refer to

_protocol

3.

Non-staining

gaps at the telomeric

_region

The results are shown in

_figure

la and lb. The minimum

_expression

value considered

was

4%

of the total observed

cells,

and

only

one animal

_presented

an

_expression

percentage

below this.

_Among

the

_{remaining 20,}

the _gaps were

_present

at a

frequency

of

30-66%

of the

_cells,

with a mean value of 48.3 t

2.1%.

Cells with more than one _gap occurred at a

_frequency

_ranging

from

0-38%

of the

_total,

with a mean value of 10.5 f

2.3%

_{(table I).}

After

destaining

and

subsequent

G-banding

the autosome

_pair

number 5 could be identified as the main carrier of _gaps

(fig

_2a,

2b and

2c).

In 18 animals

(90%

of

the

_20),

gaps on

homologous

chromosome 5 could be observed on this

pair

(fig

2a

and

_2b).

..

Chromatidic breaks

The occurrence of chromatidic breaks

_ranged

from

0%

(breaks

were detected

_only

in ten

_animals)

to

_4%,

with a mean value of 1.5 f

0.4%

(table I).

Unlike the gaps,

the break locations were detected in different

_regions

and chromosome

_pairs,

and

these

_ruptures

were observed in

only

one chromatid in all the

_analyzed

cases

_(fig

3a and

_3b).

Methodologies

used

The three variants described were useful for

_detecting

_gaps and break induction.

Considering

the ANOVA test

_performed,

treatment 3 showed

significant

differences

The

_{proliferation}

cell rate was lower when

_protocol

2 was

_employed.

For this _reason,

another

_comparison

test between

_protocols

1 and 3 was carried out

_using

18 animals

(fig 5).

The ANOVA test did not show

_{significant differences,}

but a

slight

increase

in _gap

_expression

in treatment 3 could be detected in both

_comparison

tests

_(fig

4

and

_5).

DISCUSSION

From a

morphological

point

of

view,

the two

types

of lesions found can be

con-sidered as different chromosomal

fragility

_expression

forms in the

_goat

karyotype.

The

_{high frequency}

of _gaps and the

_constancy

of their location are two features in

agreement

with the

descriptions

of other

species

whose gaps have been the most

frequent

expression

of chromosome

_{fragility. Matejka}

et al

₍₁₉₉₀₎

reported

a mean

value of

40%

expression

for a BrdU sensitive

_fragile

site located at the

_eighth

_pair

in

_sheep,

and

_Gripenberg

et al

₍₁₉₉₁₎

detected up to

86%

expression

in a fra.X. of deer. In humans the

_fra.12q24.2

and

_fra.lOq25

are both

fragile

sites with a

high

et

_al,

_1985).

These

_high

_expression

values are in

_agreement

with the mean

_expression

observed

_by

us, and reinforce the fact that our results _may

_represent

a common

folate-sensitive

_fragile

_site,

which shows a chromosome _gap as its form of

_expression

in cell culture.

Furthermore,

the three

_protocol

modifications used were useful for _gap induction.

Even

_though

some of the

_drugs

used

_might

have enhanced the

_expression,

it can be

pointed

out that the absence of folic acid in the culture medium was the main factor

detected

_only

when methotrexate was added 24 h before

_harvesting

_(a

difference was

observed

_mainly

when a

_comparison

was made between treatments 1 and

_3).

If the

effect

_produced

_{by amethopterin}

on the condensation and chromosome

_elongation

is considered

_(Laird

et

_al,

_1987),

an increase of the

_degree

of

_stretching

at the

fragile

region,

and

_consequently

a clearer observation on

_{optical microscopy,}

could

be a

logical explanation

for the observed

high

level of

expression

when this

drug

is

added.

Even

_though

in some animals

_(three),

different chromosome

_pairs

were detected

to be telomeric gap

carriers,

their

_expression

levels were

<4%

of the cells and no

chromosome

_repetitive

location was

_{confirmed; only}

in the

_pair

5 were the _gap

location and

_frequency

_expression

values

_{good enough}

to be considered a

_possible

expression

of chromosomal

_fragility.

One

_important

_{fact is that the gaps}

_overlap

the telomeric nucleolar

_{organization region}

_(NOR)

in this

_pair.

_Consequently

it is necessary to assume that any

stretching

of the NOR

region

could

provoke

a variable

percentage

of

_{false-positive fragile}

detection. In this work no

_{triple staining}

(Giemsa-G-Banding-NORs)

was

_employed,

and it is difficult to estimate what

_percentage

of

gaps

might

be

false-positive

due to active NOR

_regions

_being

detected as

_fragile

sites,

or indeed if all the

fragile

sites described here are

_only

NOR

stretchings.

There

is some evidence that the latter is not the case.

_First,

telomeric gaps were detected

only

in one

(pair 5)

of the five chromosome carriers of NORs in

_goat

(according

to

Di Meo et

_al,

₁₉₉₁₎

_{pairs 2, 3, 4,}

5 and 28 are NORs carriers in

goats.

Secondly,

the gaps were not limited to the telomere

_only,

the _gap

_region

_involving

all the telomeric

_positive

R or

negative

G bands of the carrier chromosome. The _gaps are

bigger

than the NOR

_region

detected with normal AG-NOR

_(silver

_staining

_NOR)

staining.

These features indicate that it is not valid to assume that all the _gaps are

extensions of the

_NORs,

_though

a

_percentage

of

false-positives

due to this cannot

be excluded.

Telomeric Structural

_Changes

_(TSC)

have been indicated as another source of error when human telomeric

_fragile

sites are

_analyzed

_(Butler

et

al,

1990).

These

TSC are chromosomal lesions which can be detected as

fragile

sites and the authors

mentioned that about

10%

of

_positive

detection in human fra.X is due to this kind of alteration.

_According

to this it would be necessary to take into account another

variable

_percentage

of error in the results observed.

The

_expression

rate and the different

_positions

of the breaks are

_subjects

for

discussion. The breaks were not found in the control cultures and

_{they presented}

morphological

features similar to some of the

_fragile

sites described in humans

(isochromatidic

breaks as

_{reported by}

Sutherland et

_{al, 1985,}

and

_Sutherland,

_1991).

On the other

_hand,

in no case did the break

frequency

raise the minimum level of

expression

estimated in this work

_(4%)

and

_only

eleven animals

_expressed

these

breaks with no

_repetition

established in the

_position

_(a

variation was noted in the

location within each chromosome and between chromosome

_pairs).

Taking

into account that the main source of variation for the

_fragile

_expression

is related to the induction

_methodology

_(as

described

_by

Fisch et

_al,

1991 for the

fra.X

syndrome

in

_humans),

the

_possibility

that the methods used here caused a

CONCLUSIONS

Although

this is a

preliminary study,

some conclusions may be drawn. The

ex-pression

of chromosomal

_fragility

and its induction in

_goat

_karyotype

are similar

to observations in related and unrelated

_{species. Furthermore,}

_gaps seem to be the main form of

expression.

Even

_though

a combination of FdU and a low folate

medium seems to be the

_only

condition _necessary for

_induction,

some

_drugs

like

amethopterin

and

_thymidine

are

_important

for

_improving

_fragility

_expression

in this

karyotype.

A telomeric _gap could be detected

_{successfully only}

in

_pair

_5;

_according

to its

expression

frequency

and culture conditions it could be classified as a virtual

folate-sensitive

_fragile

site. Further

_study

is needed of the sources of error which can lead

to a

significant

_percentage

of

false-positive

results

(a

combination of Telomeric

Structural

_Changes

and

_NORs).

A

_study

of its

_genetic

inheritance is also

_required

to reach a definitive conclusion.

It is not

_possible

to draw a conclusion about the chromatidic breaks detected.

The low

_frequency

of

_expression

and the variation in location on different chro-mosome

_pairs

indicate that different induction methods are _necessary for a better

understanding

of the nature of their

_expression.

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