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Chromosome analysis of bovine oocytes cultured in vitro
Br Yadav, Wa King, Kp Xu, Jw Pollard, L Plante
To cite this version:
Original
article
Chromosome
analysis
of bovine
oocytes
cultured
in
vitro
BR
Yadav*
WA
King*
KP Xu JW
Pollard,
L Plante
University of Guelph,
OntarioVeterinary College, De
P
artment
of
BiomedicalSciences,
Guel
P
h,
OntarioCanada,
NlG 2W1(Received
18July 1990; accepted
4April
1991)
Summary -
Oocytes
(n
=836)
collected fromslaughtered
cows of unknownreproductive
background
were matured in vitro andsubjected
to chromosomeanalysis.
Inall,
744(89%)
resumed meiosis and 603
(72%)
reachedmetaphase
II stage. Thenon-disjunction
rate anddisploidy
rate was 5.8% and10.7%, respectively.
The incidence ofnon-disjunction
in in vitro matured bovine oocytes is similar to thatreported
for other domesticspecies.
oocytes
/
meiosis/
non-disjunction/
chromosome/
bovineRésumé - Analyse chromosomique d’ovocytes bovins cultivés in vitro. Les ovocytes
utilisés lors de cette étude
furent prélevés
sur des ovaires de vaches de statutreproductif
inconnu. La maturation de ces ovocytes(n
=836)
fut effectuée
in vitro et suivie de leurfixation
en vue d’uneanalyse chromosomique.
Autotal,
744(89%)
présentèrent
unereprise
de méiose et 603(72%)
atteignirent
le stade demétaphase
II. Les taux denon-disjonction
et de
diploïdie
étaient de5,8%
et10,7%
respectivement.
L’incidence de lanon-disjonction
chez les ovocytes bovins maturés in vitro est similaire à cellesdéjà signalées
chez d’autresespèces domestiques.
ovocytes
/
méiose/
non-disjonction/
chromosome/
bovinINTRODUCTION
Since observations of the chromosomes of domestic cattle were first
reported by
Krallinger
in1927,
extensive studies haveyielded
a wealth of informationpertaining
to the normal
karyotype.
Inaddition,
alarge
number of individuals with abnormalkaryotypes involving
anomalies of chromosome structure and number have beenreported
(for
review seePopescu,
1989).
*
Present address :
NDRI,
National Institute of AnimalGenetics/National
Bureau of Animal GeneticResources,
NDRICampus, Karnal-Haryana
132001, India**
The
study
of thepost-synaptic
stages
of meiosisprovides
information on thechromosome
composition
ofgametes
andgives
apreview
of theembryos
thatmight
arise from them. In
bulls,
the incidence of abnormal second meioticmetaphase
has been studied(Gustavsson,
1969; Logue
andHarvey, 1978; Popescu,
1978).
In cows, thechronological
and themorphological
aspects
ofoocyte
maturation have been established(Jagiello
etal, 1974,
King
etal, 1986;
Suss etal,
1988),
however,
there isless information on the chromosomal status of the
maturing
oocyte.
With the recentdevelopment
of reliabletechniques
for the in vitro maturation and fertilization of bovineoocytes
(Leibfreid-Rutledge
etal,
1989)
large
numbers ofoocyte
which leadto
embryos
of knownkaryotype
anddevelopmental potential
can beproduced
andanalyzed.
In this paper we
report
our observations on the chromosomalcomposition
ofbovine
oocytes
matured in vitro. Theoocytes
used for thisstudy
were obtained inconjunction
with in vitro fertilization and culture of bovineoocytes
and served ascontrol observations for the
oocyte
maturationprocedure
in those studies.MATERIALS AND METHODS
The
oocytes
used for thisstudy
wereprepared by
the method describedby
Xuet al
(1987)
as follows : ovaries from cows of unknownreproductive
historieswere collected from a local abattoir. The surface of the ovaries was slashed
with a razor blade and rinsed in Hams F-10
supplemented
with2%
estrus cowserum,
heparin
(2
iu/ml)
Hepes
buffer(10
mNI)
and antibiotics(100
iupenicillin,
100 pg
streptomycin/ml).
Thecumulus-oocytes complexes
(COC)
were recoveredfrom this
collecting
medium and transferred in groups of 30-50 to 1.0 ml of Hams F-10supplemented
with estrus cow serum(20%)
and antibiotics. COC cultureswere incubated at 39°C in a humid
5%
atmosphere
CO
z
for 26 to 28 h. COC werethen removed from the
cultures,
the cumulus cells dissociatedby
mechanical forces and theoocytes
were fixed on slides aspreviously
described(King
etal,
1979).
The slides were examined after
staining
with4%
Giemsa for 4 min and thestage
of nuclear maturation
assigned
aspreviously
described(King
etal,
1986).
The chromosomes at meioticmetaphase
I(NII)
and meoiticmetaphase
II(MII)
werecounted at 1 000 x
magnification.
The rate ofnon-disjunction
(ND)
was calculatedaccording
to the formula used for bulls(Logue
andHarvey,
1978) :
RESULTS
A total of 836 oocytes were fixed for chromosome
analysis.
Thestage
of nuclear maturation could be determined in 744(89%)
oocytes
of which 23(2.6%)
werediplotene/diakinesis
stage,
118(14.1%)
were MI and 603(72.1%)
were MII. Theto resume meiosis. The distribution of the number of
chromosomes,
MI bivalentsand MII
univalents,
of the 389preparations
suitable forcounting
is summarized in table I. Of the 308 MIIspreads
suitable forcounting,
13(4.2%)
wereaneuploid
(4
hypohaploid
and 9hyperhaploid)
and 33(10.7%)
werediploid.
Thenon-disjunction
rate, calculatedaccording
to the ND formulaabove,
was 0.058 or5.8%.
Among
the 295oocytes
at the MIIstage
for which exact chromosome countscould not be
made,
a further 33(11.2%)
werethought
to bediploid.
Chromosomes of thepolar
body
were observed in 67oocytes
although they
were notpossible
toanalyze
due to contraction andclumping.
DISCUSSION
The
high
rate of nuclear maturation observed in thisstudy
(81%,
603/744)
isconsistent with other studies on the in vitro
resumption
of meiosis(eg
King
etal, 1986;
Suss etal,
1988).
Since the first observation thatoocytes
spontaneously
resume meiosis once removed from the follicle and cultured in vitro
(Pincus
andEnzmann,
1935),
it has beenrecognized
that nuclear maturation is not synonymouswith maturation and
acquisition
of fertilizationpotential
(First
etal,
1988).
IVF data show the rates of fertilization anddevelopment
are lower than those of nuclearmaturation
(Leibfried-Rutledge
etal,
1989).
Forexample,
in studiesparallel
tothis,
for which some of the
present oocytes
served ascontrols,
the rates of fertilization(penetration),
cleavage
anddevelopment
to themorula/blastocyst
stage
were71%,
57%
and28%, respectively
(Xu,
unpublished
observations).
Thisclearly
reinforces the observation that not alloocytes
which reach MIIstage
in vitro arecapable
ofbecoming
fertilized anddeveloping
into viableembryos.
It hasyet
to be determined ifchromosomally
abnormal bovineoocytes
are lesslikely
to be fertilized than normal ones. Inhumans,
up to two-thirds ofoocytes
which fail to cleave after IVFattempts
have beenreported
to bechromosomally
abnormal(eg
Wramsby
and
Fredga,
1987;
Ma etal, 1989;
Papadopoulos
etal,
1989).
However,
it must be remembered thatmorphological
as well as chromosomal abnormalities have beenChromosomal
analysis
ofoocytes
clearly
has anadvantage
over that ofsper-matocytes
in thatoocytes
can be isolated andprepared individually
eachcon-taining
at most 2metaphases. However,
one technicaldifficulty
common to bothoocytes
andspermatocytes
is chromosomal lossduring
fixation and slide prepara-tion.Non-disjunction
during
the first meiotic division isexpected
to lead toequal
portions
ofhyperhaploid
andhypohaploid
MIIspreads.
Expression
of the rate ofnon-disjunction
as twice the incidence ofhyperhaploid
MIIspreads
compensates
for thepotential
false bias towardshyperhaploidy.
However,
in thepresent
study
technical loss of chromosomes appears to not be asignificant
factor ashyperhap-loids
actually
out-numberedhypohaploids.
The incidence ofnon-disjunction
in thepresent
study
(5.8%)
is similar to that of horseoocytes
(5.5%;
King
etal,
1990)
andpig
oocytes
(3.4%;
McGaughey
andPolge,
1971).
The10.7%
incidence ofdiploid
MII ishigher
thanpreviously reported
in cattleoocytes
(2.9%; Jageillo
etal,
1974)
or horse
oocyte
(2.7%;
King
etal,
1990)
although
lower than inpig
oocytes
(12.2%;
McGaughey
andPolge,
1971).
Inbulls,
the incidence ofnon-disjunction
has beenreported
as2.8%
(Logue
andHarvey,
1978).
In astudy
involving
a Robertsoniantranslocation a
7.7%
incidence ofdiploid
MII was observed(Gustavsson, 1969).
The similar incidence ofaneuploidy
andpolyploidy
in otherspecies
suggests
a base-lineincidence of
non-disjunction
common to these domesticspecies.
Fertilization of the
oocytes
withhypohaploid, hyperhaploid
ordiploid
MIIconfigurations by
spermatozoon
with a normal chromosomecomplement
would leadto the formation of an
aneuploid
orpolyploid embryo.
Chromosomeanalysis
ofbovine
embryos
produced
by
fertilization in vivo andranging
in age from 1 to 18 d has shown an overall incidence ofrecognizable
abnormalities of ?10%.
Included inthis group are abnormalities such as
mixoploidy
which arethought
to occur aspost-meiotic events
during
fertilization andcleavage
(King, 1990).
The mostfrequently
observed
abnormality
inembryos produced by
IVF has beentriploidy
(10%;
Iwasakiet
al, 1989;
7%;
King
etal,
1988).
The incidence oftriploidy
in bovineembryos
dueto fertilization of a
diploid
oocyte
(digyni)
is not knownalthough
the observations of thisstudy
suggest
that it could be ashigh
as10.7%.
Inparallel
studies theincidence of
dispermic
orpolyspermic fertilization,
as evidencedby
the presenceof 2 or more male
pronuclei,
was5%
(Xu,
unpublished
observations).
Studies ontriploid
humanspontaneous
abortusessuggest
that agreater
proportion
(85%)
arise,due
todiandry
(fertilization
by
2spermatozoa
orby
asingle
diploid
spermatozoon)
than from
digyni
(15%;
Jacob etal,
1982).
Only
a fewexamples
of pureaneuploidy
have been observed in bovineembryos
produced
by
fertilization in vivo(King, 1990).
Less than1%
ofembryos produced
by
IVF were observed to beaneuploid
(Iwasaki
etal,
1989;
King
etal,
unpublished
data collected in
parallel
to thepresent
study).
This incidence issubstantially
lower than whatmight
beextrapolated
from thenon-disjunction
rates observed inoocytes
(5.8%;
present
study)
andspermatocytes
(2.5%;
Logue
etal,
1978).
However,
it has been noted that there is aprogressive
elimination ofembryos
with abnormalkaryotype throughout
early
stages
ofdevelopment
(King, 1990).
Furthermore,
notall fertilized
oocytes
arecapable
ofcompleting
even the firstcleavage
division(Xu
et
al,
1987).
Hence,
aportion
ofaneuploid embryos
may go undetected in thezygote
orearly embryo.
Thepossibility
thathypo-
orhyperhaploid
oocytes
are lesslikely
In cattle .::
90%
of ovulated ova arefertilized,
however, calving
rates arecloser to
50%
withembryo
lossmaking
up the difference between fertilization andcalving
rates(Diskin
andSreenan,
1980).
While the incidence of chromosome abnormalities is notyet
well established in bovineembryos,
it is clear thatthey
are a factorcontributing
toembryonic
loss. A smallportion
ofembryos
with abnormalkaryotype, aneuploidy
of the sex chromosomes(eg
Pinheiroal,
1987)
or on rareoccasions autosomal
trisomy
( eg
Mayr
etal,
1985),
do survive to term. Noexamples
of live-borntriploid
cattle have beenreported. Interestingly,
gametes
that couldproduce aneuploid
individuals are the leastfrequently
observedproduct
of meiosis.ACKNOWLEDGMENTS
The financial support of NSERC
Canada,
Agriculture
Canada,
The Canadian Association of Animal Breeders and SEMEX Canada isgratefully acknowledged.
BRY received ascholarship
from theDepartment
ofBiotechnology, Ministry
of Science andTechnology,
Government of India. The technicalhelp
of E St John isappreciated.
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