Analysis of the 1/20 translocation in ram spermatozoa by flow cytometry

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Analysis of the 1/20 translocation in ram spermatozoa

by flow cytometry

H Lewalski, Fj Otto, T Kranert, B Glahn-Luft, R Wassmuth

To cite this version:

Analysis

of the

_1/20

translocation

in

ram

_spermatozoa

by

flow

cytometry

H

Lewalski

1

FJ

Otto

2

T Kranert

3

B

Glahn-Luft

R Wassmuth

1

Institut

_für

Tierzucht und

_{Haustiergenetik}

der

Justus-Liebig-Universitaet,

Gießen;

2

Abteilung für Experimentelle Tumorforschung

der Fachklinik Hornheide, Muenster;

3

Strahlenzentrum der

Justus-Liebig-Universitaet,

Gießen, Germany

(Proceedings

of the 9th European

Colloquium

on

_Cytogenetics

of Domestic

_Animals;

Toulouse-Auzeville,

10-13

_July

₁₉₉₀₎

unbalanced ram _{spermatozoa /} flow _cytometry

/ 1/20

translocation

INTRODUCTION

Structural

_{rearrangements}

of

_chromosomes,

_including

translocations can have ad-verse effects on

_fertility

in humans

_(Daniel,

_1988;

_Lange

et

_al,

₁₉₉₀₎

and animals

(Gustavsson,

1980;

Eldridge,

1985; Daniel,

1988).

The formation of unbalanced

ga-metes

_during

meiosis in

_heterozygous

translocation carriers may cause unbalanced

embryos

which fail to survive to the

_implantation

_stage

_{of pregnancy} or die in later

stages

of

_development.

In

_sheep,

two different translocations are known. Bru6re et al

₍₁₉₇₄₎

observed 3

types of Robertsonian translocations

_{designated Massey}

I

_(5/26),

II

_(8/11)

and III

(7/25),

but

_reproduction

of carriers was not

_negatively

affected

_by

the translocations

(Bru!re,

1975; Long, 1977, 1978;

Bru6re and

Ellis,

1979).

Long

(1977)

and Bru6re

et al

₍₁₉₈₁₎

concluded that a selection mechanism in

_heterozygous

translocation-carrying

rams

_{during spermatogenesis}

eliminates unbalanced

_secondary

spermato-cytes.

On the other

_hand,

carriers of the

_1/20

translocation showed a reduction

in

_fertility

_(Glahn-Luft

and

Wassmuth, 1980,

1985).

These results led to the

assumption

that

_heterozygous

_1/20

translocation carriers

_produce

unbalanced

gametes

causing

early embryonic mortality.

A method to confirm the existence of unbalanced

_gametes

is the measurement of their DNA content. Flow

cytometry

has been used to measure the relative DNA

con-tent of X and Y

_{chromosome-bearing}

sperm

populations

(Otto

et

al, 1979;

Garner

et

_al,

_1983,

_Johnson,

₁₉₈₇₎

as well as to demonstrate chromosomal

_{rearrangements}

MATERIALS AND METHODS

Freshly

ejaculated

semen was collected from

_heterozygous

_1/20

translocation

car-riers as well as

_presumed

non-carriers of various

_sheep

breeds. The

samples

were

prepared

for flow

_cytometric

_analysis

as described

_by

Otto et al

_(1979).

_Suspensions

of stained sperm nuclei were measured

_using

a PAS II flow

_cytometer

(Partec

AG,

Arlesheim,

Switzerland).

RESULTS AND DISCUSSION

The

_histogram

of

_spermatozoa

from a normal ram shows a distinct

_separation

of X and Y

_{chromosome-bearing}

_spermatozoa

_(fig

_1).

The

_analysis

revealed a difference

of

4.5%

in the DNA content _{of gonosomes and} a ratio of

48%

Y and

52%

X

spermatozoa.

The

_1/20

translocation involves the

_breakage

of the distal _part of the short arm of chromosome 1 and its transfer to the distal

_part

of chromosome 20

_(Glahn-Luft

and

Wassmuth,

1984).

Considering

alternate and

_adjacent

chromosome 1

_segregation

at

diakinesis, heterozygous

1/20

translocation-carrying

rams are

_expected

to

_produce

increased DNA content. The

_resulting

ratio of the different

_spermatozoa

fractions

is

_1:2:1:1:2:1,

which

_implies

that

50%

normal or balanced and

50%

unbalanced spermatozoa are

_{ejaculated by}

_heterozygous

_1/20

_{translocation-carrying}

rams. Due

to the relative size of the translocated chromosomal

_fragment

_{(approximately}

2%)

and the

_expected

ratio of sperm

populations,

it was

_possible

to construct

an

_empirical

_histogram

_using

individual Gaussian curves. The

_histogram

shows 5 instead of the

_expected

6

peaks

(fig 2). This is

due to the size of the translocated

segment,

since Y _sperm with an increased and X _sperm with a reduced DNA content

merge between the 2 main

peaks

of X and Y

_spermatozoa

which contain normal and balanced sperm.

In

_agreement

with the calculated

_histogram,

the flow

_{cytometric analysis}

of

ejaculated

sperm from the

1/20 translocation-carrying

ram 7402 also revealed

5

_peaks

_{(fig 3),}

_{demonstrating}

the existence of unbalanced

_spermatozoa.

The ratio of individual _sperm fractions as well as the

_positions

of the

_peaks

are in concordance

with our theoretical calculations.

Using

flow

_cytometry,

the

_production

of unbalanced

_spermatozoa

_by

heterozy-gous

1/20

translocation-carrying

rams was confirmed. To determine whether this

indeed has a

_negative

effect on

_reproduction

_through

the formation of unbalanced

REFERENCES

Bru6re AN

₍₁₉₇₅₎

Further evidence of normal

fertility

and the formation of balanced

gametes in

_sheep

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_Reprod

Fertil

45, 323-331

Bru6re

_AN,

Ellis PM

₍₁₉₇₉₎

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with

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J

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Bruère

_AN,

Zartman

_DL,

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HM

₍₁₉₇₄₎

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Cytogenet

Cell Genet 13, 479-488

Bru6re

AN,

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IS,

Henderson LM

₍₁₉₈₁₎

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₍₁₉₈₈₎

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FE

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Wassmuth R

₍₁₉₈₄₎

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₍₁₉₈₇₎

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₍₁₉₇₉₎

Flow cytometry