Note: Descriptions are shown in the official language in which they were submitted.
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ISOLATING A CYTOPLASMIC FRACTION WITHOUT IMPAIRING THE VIABILITY OF OOCYTES
AND EMBRYONIC
CELLS
TECHNICAL FIELD
The present invention relates to a method of isolating
a cytoplasmic fraction from an oocyte which does not
impair the capacity of the oocyte to be fertilized. The
invention also relates to a method of isolating a
cytoplasmic fraction from an embryonic cell which does not
impair the developmental potential of the cell.
BACKGROUND ART
Methods for the analysis of the oocyte cytoplasm have
necessitated the destruction of the oocyte. Consequently,
although the information retrieved in the analysis may be
of particular interest, the investigator is unable to
further study the oocyte as a complete cellular unit in
the context of the information retrieved from the
analysis. Further, the oocyte cannot subsequently be
fertilized and allowed to develop into an embryo, fetus
and infant.
There is a need for a method which allows the study of
cytoplasmic mechanisms in the oocyte which does not impair
the capacity of the oocyte to be fertilized. There is
also a need for a method which allows the study of
cytoplasmic mechanisms in an embryonic cell which does not
impair the developmental potential of the cell. These
methods would be useful for studying those mechanisms in
an oocyte or embryonic cell which are mediated by
mictochondria and which are the cause of, or are suspected
of causing, or are associated with, dysfunction or
disease, either in the oocyte or embryonic cell, or in
progeny descended from the fertilized oocyte, or the
embryonic cell.
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DESCRIPTION OF THE INVENTION
The present invention seeks to address these needs and
in a first aspect provides a method of isolating a
cytoplasmic fraction from an oocyte which does not impair
the capacity of the oocyte to be fertilized, the method
including the step of releasing a cytoplasmic fraction
from the oocyte. The volume of cytoplasmic fraction which
is released from the oocyte is about 5% of the volume of
the oocyte. Preferably the volume is about 2% of the
volume of the oocyte.
In an embodiment of the first aspect of the invention,
the method includes the following steps:
a) inserting releasing means into the oocyte;
b) drawing a cytoplasmic fraction which is about 50
of the volume of the oocyte into the releasing
means; and
c) withdrawing the releasing means from the oocyte
so that the fraction is isolated in the releasing
means .
The volume of the cytoplasmic fraction drawn into the
releasing means is typically less than 10 pL and
preferably 8 pL. Preferably, the releasing means includes
at least an ICSI pipette and preferably the cytoplasmic
fraction is drawn approximately 100~.~.m into the pipette.
The drawing of the cytoplasmic fraction from the
oocyte into the releasing means typically forms an
extrusion of cytoplasmic contents between the releasing
means and the oocyte. The cytoplasmic fraction is isolated
from the oocyte by gently stretching or shearing the
extrusion so as to separate the extrusion. Preferably the
cytoplasmic fraction is isolated by stretching the
extrusion.
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Typically, the cytoplasmic fraction contains
cytoplasmic organelles, and includes or consists of a
sample of the oocyte's mitochondria and mitochondria)
products.
Mitochondria located in an oocyte are a template from
which all mitochondria in the progeny descended from the
fertilized oocyte are derived. The present inventors
recognised that a method for isolating a cytoplasmic
fraction from an oocyte, without impairing the capacity of
the oocyte to be fertilized, would allow the study of the
relationship between the integrity of the mitochondria)
genome and the function of both the oocyte and the progeny
descended from the fertilized oocyte.
Thus, in a second aspect, the invention relates to a
method of analysing the mitochondria) genome of
mitochondria located in an oocyte which does not impair
the capacity of the oocyte to be fertilized, the method
including the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention;
b) analysing the mitochondria) genome of the
mitochondria in the fraction.
Mutation of the mitochondria) genome causes, or at
least is associated with, dysfunction or disease. For
example, the nucleotide sequence deletion from nucleotide
position number 8470 to 13,446 of the mitochondria)
genome, the so called "5kb common deletion", is understood
to be associated with Kearns-Sayre syndrome (KSS) and
chronic progressive external opthalmoplegia (CPEO). Other
disease causing deletions, which may or may not be
observed with the common deletion, include a 7.4 kb
deletion and 10.4 kb deletion/insertion in the
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mitochondrial genome of brain and heart,. as well as
various point mutations. The deletion is also observed in
human tissue including skeletal muscle, heart, brain,
oocytes, leukocytes, retina and ovaries.
Over 40 pathogenic point mutations of the
mitochondrial genome can be associated with a broad
spectrum of degenerative diseases involving the central
nervous system, heart, muscle, endocrine system, kidney
and liver. Diseases associated with point mutations
include Leigh Syndrome, MELAS (mitochonrdial
encephalomyopathy, lactic acidosis and stroke like
episodes), MERRF (myoclonus epilepsy with ragged-red
fibres), NARP (neruopathy, ataxia and retinitis
pigmentosa) and LHON (Leber hereditary optic neuropathy).
A method of isolating a cytoplasmic fraction from an
oocyte without impairing the capacity of the oocyte to be
fertilized is useful for detecting nucleotide sequence
mutations in the mitochondrial genome, enabling the study
of a nucleotide sequence, polymorphism or mutation in the
context of the functional integrity of both the oocyte and
the progeny descended from the fertilized oocyte.
Thus, in a third aspect, the invention provides a
method of detecting a nucleotide sequence, polymorphism or
mutation in the mitochondrial genome of mitochondria
located in an oocyte which does not impair the capacity of
the oocyte to be fertilized, the method including the
following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention; and
b) analysing the nucleotide sequence of the
mitochondrial genome of the mitochondria in the
cytoplasmic fraction for the presence of a
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nucleotide sequence, polymorphism or mutation in
the mitochondria) genome.
As mitochondria) genome mutations which cause, or are
associated with, disease or dysfunction, including point
mutations, are almost exclusively maternally inherited, a
method of isolating a cytoplasmic fraction without
impairing the capacity of the oocyte to be fertilized is
useful for predicting whether the progeny descended from a
fertilized oocyte will or will not contain a nucleotide
sequence, polymorphism or mutation of the mitochondria)
genome which causes, or is suspected of causing, or is
associated with, disease or dysfunction.
Thus in a fourth aspect, the invention provides a
method for predicting whether the progeny descended from a
fertilized oocyte will contain a nucleotide sequence,
polymorphism or mutation in a mitochondria) genome which
causes, or is suspected of causing, or is associated with,
a disease or dysfunction, wherein the method does not
impair the capacity of the oocyte to be fertilized, the
method including the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention; and
b) analysing the mitochondria) genome of the
mitochondria in the fraction for the presence of
the nucleotide sequence, polymorphism or
mutation in the mitochondria) genome;
wherein the presence of the nucleotide sequence,
polymorphism or mutation indicates a likelihood that the
progeny descended from the fertilized oocyte will contain
the nucleotide sequence, polymorphism or mutation.
The mere existence of a nucleotide sequence,
polymorphism or mutation in the mitochondria) genome of
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mitochondria in an oocyte, which is known to be associated
with, or cause, or is suspected of causing, disease or
dysfunction, may not be sufficient to mediate the disease
or dysfunction in the oocyte itself, or the progeny
descended from the fertilized oocyte. Indeed, there
appears to be at least an additional factor which
contributes to the likelihood of, and/or the severity of
the disease or dysfunction in the oocyte containing the
nucleotide sequence, polymorphism or mutation, or the
progeny descended from the fertilized oocyte. That is, it
is likely that the actual proportion, or "threshold
level", of mitochondria which contain the particular
nucleotide sequence, polymorphism or mutation in the
mitochondrial genome in the oocyte will contribute to the
likelihood and/or severity of disease or dysfunction in
the oocyte, or progeny descended from the fertilized
oocyte. In particular, and in contrast with the nuclear
genome, there are of the order of at least 100,000 copies
of a mitochondrial genome in an oocyte. "Heteroplasmy" is
observed when an oocyte contains more than one species of
mitochondrial genome. An oocyte, and indeed the tissues of
the progeny descended from the fertilized oocyte, become
"heteroplasmic" when a nucleotide sequence, polymorphism
or mutation is introduced into a cell which, before the
introduction, contained a single species of mitochondrial
genome. With regard to the nucleotide sequences,
polymorphisms and mutations of the mitochondrial genome
which cause, or are suspected of causing, or are
associated with, a disease or dysfunction, it is generally
recognised that when a particular level of heteroplasmy is
surpassed, the manifestations of the disease or
dysfunction are sooner or later observed.
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Based on the observation that mitochondria in the
mouse oocyte migrate to specific regions of the oocyte
cytoplasm, in particular, the perinuclear region, at
specific stages of meiosis, to obtain a sample which is
representative of all mitochondrial genomes in the human
oocyte, a method for determining the degree of
heteroplasmy in a human oocyte is typically applied at
specific stages of meiosis. A method of isolating
cytoplasmic fractions which does not impair the capacity
of the oocyte to be fertilized is useful for determining
the level of heteroplasmy in the oocyte, and for
predicting the average level or likely range of
heteroplasmy in tissues of the progeny descended from the
fertilized oocyte.
Thus, in a fifth aspect, the invention provides a
method of determining the level of heteroplasmy of
mitochondrial genomes in an oocyte which does not impair
the capacity of the oocyte to be fertilized, the method
including the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention;
b) comparing the number of mitochondrial genomes in
the fraction with a nucleotide sequence,
polymorphism or mutation, with the number of
genomes without the nucleotide sequence,
polymorphism or mutation in the fraction.
As the level of heteroplasmy and the existence of a
nucleotide sequence, polymorphism or mutation which
causes, or is suspected of causing, or is associated with,
disease or dysfunction, in progeny descended from a
fertilized oocyte contribute to the likelihood and/or
severity of disease or dysfunction, the method of
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determining the level of heteroplasmy of mitochondrial
genomes in an oocyte is useful for predicting whether the
progeny descended from the fertilized oocyte are likely to
suffer from a disease or dysfunction which is caused by,
or associated with, the particular nucleotide sequence,
polymorphism or mutation, and/or the severity of the
disease or dysfunction.
Thus in a sixth aspect, the invention provides a
method of determining whether the progeny descended from a
fertilized oocyte are likely to suffer from a disease or
dysfunction caused by, or suspected of being caused by, or
associated with, a nucleotide sequence, polymorphism or
mutation in a mitochondrial genome, wherein the method
does not impair the capacity of the oocyte to be
fertilized and includes the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention;
b) analysing the mitochondrial genome of the
mitochondria in the fraction for the presence of
the nucleotide sequence, polymorphism or
mutation; and
determining that the progeny are likely to suffer from the
disease or dysfunction where the analysis of the
mitochondrial genome of mitochondria in the fraction
demonstrates that the level of heteroplasmy of
mitochondrial genomes with respect to the nucleotide
sequence, polymorphism or mutation in the oocyte is at
least the same as the level of heteroplasmy which is known
to be associated with the manifestation of the disease or
dysfunction.
As noted above, methods for the analysis of
cytoplasmic fractions from an oocyte involve the
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destruction of the oocyte. Although these methods reveal
useful information about the oocyte that is thereby
destroyed, the value of this information is confined to an
extrapolation to other oocytes which are not evaluated. As
a population of oocytes from the same individual are
typically heterogenous with respect to ooplasmic content,
there is some question as to the extent to which the
information derived from the oocyte which is destroyed can
be accurately or reliably extrapolated to other oocytes.
That is, using these methods one cannot conclude that
because an oocyte is found to have either no nucleotide
sequence, polymorphism or mutation in the mitochondrial
genome which causes, or is associated with, disease or
dysfunction in the oocyte, or the progeny of the
fertilized oocyte, or a low degree of heteroplasmy in
relation to the nucleotide sequence, polymorphsim or
mutation, that other oocytes derived from the same
individual will have the same mitochondrial genotype.
Consequently, these methods for the analysis of oocytes
are of limited use in the field of in vitro fertilization,
where it is anticipated that some oocytes derived from a
patient may contain a nucleotide sequence, polymorphism or
mutation which causes, or is suspected of causing, or is
associated with, disease or dysfunction in the progeny
descended from the fertilized oocyte.
The method of isolating a cytoplasmic fraction from an
oocyte which does not impair the capacity of the oocyte to
be fertilized is particularly useful for screening oocytes
for the presence of a nucleotide sequence, polymorphism or
mutation which causes, or is suspected of causing, or is
associated with, disease or dysfunction in the progeny
descended from the fertilized oocyte, prior to
fertilization.
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Thus in a seventh aspect, the invention provides a
method of screening an oocyte for the presence of a
nucleotide sequence, polymorphism or mutation in the
mitochondria) genome in the oocyte which causes, or is
suspected of causing, or is associated with, disease or
dysfunction in the progeny descended from the fertilized
oocyte, wherein~the method does not impair the capacity of
the oocyte to be fertilized, and includes the following
steps:
l0 a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention;
b) analysing the mitochondria) genome of the
mitochondria in the fraction for the presence of
the sequence, polymorphism or mutation.
Patients who present for in vitro fertilization
treatment frequently have few oocytes available for
selection for fertilization, and of the available oocytes,
it is anticipated that some of these will contain a
nucleotide sequence, polymorphism or mutation in a
mitochondria) genome which causes or is associated with a
disease or dysfunction in progeny descended from the
fertilized oocyte. Other methods for isolating a
cytoplasmic fraction from an oocyte are particularly
unsuitable for selecting oocytes for fertilization, and
could potentially result in the destruction of oocytes
which do not contain a nucleotide sequence, polymorphism
or mutation in a mitochondria) genome which causes or is
associated with a disease or dysfunction, or oocytes which
have a low level of heteroplasmy with respect to that
mutation.
The method of isolating a cytoplasmic fraction from an
oocyte which does not impair the capacity of the oocyte to
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be fertilized is particularly useful for selecting oocytes
for fertilization which do not contain a nucleotide
sequence, polymorphism or mutation which causes, or is
suspected of causing, or is associated with, disease or
dysfunction in the progeny descended from the fertilized
oocyte, prior to fertilization.
Thus in an eighth aspect, the invention relates to a
method of selecting an oocyte for fertilization which
either:
(i) does not contain a nucleotide sequence,
polymorphism or mutation in the mitochondrial
genome of mitochondria in the oocyte which
causes, or is suspected of causing, or is
associated with, a disease or dysfunction in the
progeny descended from the fertilized oocyte; or
(ii) has a level of heteroplasmy of mitochondrial
genomes with respect to the nucleotide sequence,
polymorphism or mutation which is less than the
level of heteroplasmy which is known to be
associated with the manifestation of the disease
or dysfunction;
wherein the method does not impair the capacity of the
oocyte to be fertilized, and includes the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention;
b) analysing the mitochondrial genome of the
mitochondria in the fraction; and
c) selecting the oocyte for fertilization, provided
that at least the degree of heteroplasmy of
mitochondrial genomes with respect to the
nucleotide sequence, polymorphism or mutation in
the mitochondrial genome of the oocyte, is less
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than the degree of heteroplasmy which is known to
be associated with the manifestation of the
disease or dysfunction.
The oocytes which are screened or selected in
accordance with the seventh or eighth aspect of the
invention, respectively, may be fertilized by intra
cytoplasmic sperm injection (ICSI), or by in vitro
fertilization. Preferably the oocytes are fertilized by
ICSI.
To the extent that the eighth aspect of the invention
is particularly suitable for selecting oocytes for
fertilization and can be used prior to ICSI or IVF, the
present inventors recognise that the method embodied in
the eighth aspect of the invention is an integral part of
a novel in vitro fertilization procedure.
Accordingly, in a ninth aspect, the invention provides
a method of fertilizing an oocyte, the method including
the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the oocyte according to the
method of the first aspect of the invention;
b) analysing the mitochondrial genome of the
mitochondria in the fraction for the presence of
a nucleotide sequence, polymorphism or mutation
which causes, or is suspected of causing, or is
associated with, a disease or dysfunction in
progeny descended from the fertilized oocyte;
and
c) fertilizing the oocyte, provided that the degree
of heteroplasmy of mitochondrial genomes with
respect to the nucleotide sequence, polymorphism
or mutation in the oocyte, is less than the
degree of heteroplasmy which is known to be
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associated with the manifestation of the disease
or dysfunction.
The mitochondria) genome of mitochondria which are in
the cytoplasmic fraction isolated according to the method
of the first aspect of the invention can be analysed
according to standard techniques. These techniques are
exemplified further herein and include the polymerase
chain reaction (PCR), restriction fragment length
polymorphism (RFLP) analysis, genomic hybridisation,
nucleotide sequencing and gene function detection and/or
measurement.
When determining the level of heteroplasmy of
mitochondria) genomes in the oocyte, a representative
fraction of genomes can be obtained when mitochondria are
randomly distributed in the cytoplasm. Preferably a
cytoplasmic fraction is drawn from the oocyte when the
distribution of mitochondria in the oocyte can be expected
to be random, including for example at the germinal
vesicle (GV) stage of the primary oocyte and /or at a
stage from the metaphase II stage of meiosis of the
secondary oocyte, to prior to syngamy.
As the method of isolating a cytoplasmic fraction from
an oocyte does not interfere significantly with the
metabolism of the oocyte, the present inventors recognised
that the method of isolating a cytoplasmic fraction is
useful for studying the cytoplasm of other cells, for
example embryonic cells, without impairing the
developmental potential of these cells.
Thus, in an tenth aspect, the invention provides a
method of isolating a cytoplasmic fraction from an
embryonic cell which does not impair the developmental
potential of the cell, the method including the step of
releasing a cytoplasmic fraction from the cell. The
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volume of cytoplasmic fraction which is.released from the
cell is about 5% of the volume of the cell. Preferably
the volume is about 2% of the volume of the cell.
In an embodiment of the tenth aspect of the invention,
the method includes the steps of:
a) inserting releasing means into the embryonic
cell;
b) drawing a cytoplasmic fraction which is about 50
of the volume of the embryonic cell into the
releasing means; and
c) withdrawing the releasing means from the cell so
that the fraction is isolated in the releasing
means.
The volume of the cytoplasmic fraction drawn into the
releasing means is typically less than lOpL and preferably
8pL. Preferably, the releasing means includes at least an
ICSI pipette and preferably the cytoplasmic fraction is
drawn approximately 100~1m into the pipette.
The drawing of the cytoplasmic fraction from the
embryonic cell into the releasing means typically forms an
extrusion of cytoplasmic contents between the releasing
means and the embryonic cell. The cytoplasmic fraction is
isolated from the embryonic cell by gently stretching or
shearing the extrusion so as to separate the extrusion.
Preferably the cytoplasmic fraction is isolated by
stretching the extrusion.
Typically, the cytoplasmic fraction contains
cytoplasmic organelles, and includes or consists of a
sample of the mitochondria and mitochondrial products of
the embryonic cell.
As described herein above, various deletions and point
mutations of the mitochondrial genome are known to cause,
or at least to be associated with, dysfunction or disease.
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A method of isolating a cytoplasmic fraction from an
embryonic cell which does not impair the developmental
potential of the cell is useful for detecting a nucleotide
sequence, polymorphism or mutation in the mitochondrial
genome, enabling the study of the nucleotide sequence,
polymorphism or mutation in the context of the functional
integrity of both the embryonic cell and the progeny
descended from the embryonic cell.
Thus in an eleventh aspect, the invention provides a
method of detecting a nucleotide sequence, polymorphism or
mutation in the mitochondrial genome of mitochondria
located in an embryonic cell which does not impair the
developmental potential of the cell, the method including
the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the embryonic cell according
to the method of the tenth aspect of the
invention; and
b) analysing the nucleotide sequence of the
mitochondrial genome of the mitochondria in the
cytoplasmic fraction for the presence of a
nucleotide sequence, polymorphism or mutation in
the mitochondrial genome.
The present inventors recognised that a method of
isolating a cytoplasmic fraction without impairing the
developmental potential of an embryonic cell is useful for
predicting whether the progeny descended from the
embryonic cell will or will not contain a nucleotide
sequence, polymorphism or mutation of the mitochondrial
genome which causes, or is suspected of causing, or is
associated, with disease or dysfunction.
Thus, in a twelfth aspect, the invention provides a
method for predicting whether the progeny descended from
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an embryonic cell will contain a nucleotide sequence,
polymorphism or mutation in a mitochondrial genome which
causes, or is suspected of causing, or is associated with,
a disease or dysfunction, wherein the method does not
impair the developmental potential of the cell, the method
including the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the embryonic cell according to the
method of the tenth aspect of the invention; and
b) analysing the mitochondrial genome of the
mitochondria in the fraction for the presence of the
nucleotide sequence, polymorphism or mutation;
wherein the presence of the nucleotide sequence,
polymorphism or mutation indicates a likelihood that the
progeny descended from the embryonic cell will contain the
nucleotide sequence, polymorphism or mutation.
As discussed herein above, it is likely that a
particular level of heteroplasmy must be surpassed before
the manifestations of disease or dysfunction are observed,
which are caused by, or associated with, a nucleotide
sequence, polymorphism or mutation of the mitochondrial
genome.
Thus, in a thirteenth aspect, the invention provides a
method of determining the level of heteroplasmy of
mitochondrial genomes in an embryonic cell which does not
impair the developmental potential of the cell, and which
includes the following steps
a) isolating a cytoplasmic fraction which includes
mitochondria from the embryonic cell according
to the method of the tenth aspect of the
invention; and
b) comparing the number of mitochondrial genomes in
the fraction with a nucleotide sequence,
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polymorphism or mutation, with the number of
genomes without the nucleotide sequence,
polymorphism or mutation in the fraction.
In a fourteenth aspect, the invention provides a
method of determining whether the progeny descended from
an embryonic cell are likely to suffer from a disease or
dysfunction caused by, or suspected of being caused by or
associated with, a nucleotide sequence, polymorphism or
mutation in a mitochondria) genome, wherein the method
does not impair the developmental potential of the cell,
and includes the following steps:
a) isolating a cytoplasmic fraction which includes
mitochondria from the embryonic cell according to the
method of the tenth aspect of the invention; and
b) analysing the mitochondria) genome of the
mitochondria in the fraction for the presence of the
nucleotide sequence, polymorphism or mutation;
determining that the progeny are likely to suffer from the
disease or dysfunction where the analysis of the
mitochondria) genome of mitochondria in the fraction
demonstrates that the level of heteroplasmy of
mitochondria) genomes with respect to the nucleotide
sequence, polymorphism or mutation in the embryonic cell
is at least the same as the level of heteroplasmy which is
known to be associated with the manifestation of the
disease or dysfunction.
Prior to commencing the embryo transfer procedure it
is important to select
(i) an embryo which does not contain a nucleotide
sequence, polymorphism or mutation of the
mitochondria) genome which causes, or is
suspected of causing, or is associated with,
disease or dysfunction, and/or
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(ii) an embryo which has a low level of heteroplasmy
with respect to that nucleotide sequence,
polymorphism or mutation.
The method of isolating a cytoplasmic fraction from
an embryonic cell which does not impair the developmental
potential of the cell is particularly useful for selecting
an embryo for embryo transfer which does not contain a
nucleotide sequence, polymorphism or mutation which causes
or is associated with disease or dysfunction in the
progeny descended from the embryonic cell, prior to embryo
transfer.
Thus, in a fifteenth aspect, the invention provides a
method of selecting an embryo for embryo transfer, which
either:
(i) does not contain a nucleotide sequence,
polymorphism or mutation in the mitochondrial
genome of mitochondria in an embryonic cell
derived from the embryo, which causes, or is
suspected of causing, or is associated with, a
disease or dysfunction in the progeny descended
from the embryonic cell or embryo; or
(ii) has a level of heteroplasmy of mitochondrial
genomes with respect to the nucleotide sequence,
polymorphism or mutation which is less than the
level of heteroplasmy which is known to be
associated with the manifestation of the disease
or dysfunction;
wherein the method does not impair the developmental
potential of the embryo, and includes the following
steps
a) isolating a cytoplasmic fraction which includes
mitochondria from the embryonic cell according to the
method of the tenth aspect of the invention; and
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b) analysing the mitochondrial genome of the
mitochondria in the fraction; and
c) selecting the embryo for embryo transfer,
provided that at least the degree of heteroplasmy
of mitochondrial genomes with respect to the
nucleotide sequence, polymorphism or mutation in
the mitochondrial genome of the embryonic cell,
is less than the degree of heteroplasmy which is
known to be associated with the manifestation of
the disease or dysfunction.
In a seventeenth aspect, the invention relates to a
kit for use in the method of isolating a cytoplasmic
fraction from an oocyte, or from an embryonic cell. The
kit includes at least one nucleotide probe specific for a
nucleotide sequence, polymorphism or mutation in the
mitochondrial genome of mitochondria in an oocyte or an
embryonic cell, which causes, or is suspected of causing,
or which is associated with a disease or dysfunction in
the progeny descended from the fertilized oocyte or
embryonic cell. In one embodiment, the at least one
nucleotide probe is specific for any one of the nucleotide
sequence mutations in the mitochondrial genome shown in
Table 1.
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Table 1
Position RNA Gene Disease
721 r 12s ADPD Alzheimers/Parkinsons
1555 r 12s DEAF, weak
1606 t Val Encephalomyopathy
1642 t Val MELAS
1644 t Val Leighs
3010 r 16s longevity
3196 r 16s ADPD
3243 t Leu(UUR) MELAS
3250 t Leu(UUR) MM Mitochondria) myopathy
3251 t Leu(UUR) MM
3254 t Leu(UUR) diabetes
3256 t Leu(UUR)
3260 t Leu(UUR) HCM Hypertrophic cardiomyopathy
3271 t Leu(UUR) MELAS later onset than 3243
3288 t Leu(UUR) MM
3302 t Leu(UUR) MM
3303 t Leu(UUR) HCM/MM
3394 m ND1 LHON also implicated in long U
interval in ECG
3397 m ND1 ADPD
3460 m ND1 LHON severe
4160 m ND1 LHON+
4216 m ND1 LHON
4269 t Ile FICP Fatal infantile
cardiomyopathy plus
4274 t Ile CPEO
4317 t Ile FICP
4336 t Gln ADPD (weak)
4917 m ND2 LHON
5178 m ND2 longevity
5244 m ND2 LHON
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Position RNA Gene Disease
5521 t Trp Myopathy, late-onset
6480 m COI
6930 COI G6930A (stop
codon):multisystem
7444 m COI LHON
7445 m COI PPK Palmoplantar
keratoderma & hearing
loss
7472 t Ser Myoclonus epilepsy,
w/o RRF
7497 t Ser MERRF Myoclonus epilepsy
etc
7512 t Ser Myoclonus epilepsy,
w / o RRF
8342 t Lys Ext. ophthalmoplegia,
myopathy, w.o RRF
8344 t Lys MERRF Myoclonic epilepsy,
ragged red fibres;
lipomatosis
8356 t Lys MERRF
8414 m ATP8 longevity
8851 ATP6 Bilateral striatal
necrosis
8860 ATP6 normal A->G
8993 m ATP6 NARP/Leighs Neurogenic weakness,
ataxia, retinitis
9176 m ATP6 Bilateral striatal
necrosis
9438 m COI LHON
9804 m COI LHON
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Position RNA Gene Disease -
9997 t Gly HCM
10004 t Gly Sudden childhood death?
10010 t encephalomyopathy
10410 t Arg Alpers syndrome
11778 m ND4 LHON severe
18832 m ND4 myopathy
12258 t Leu2 (CUN) retinitis pigmentosa,
sensorineural hearing
loss
12300 t Leu2 (CUN) Mutation of anticodon
protects against UUR
reading
12320 t Leu2 (CUN) Increased in age in
one patient with
myopathy
13708 m ND5 LHON
13730 m ND5 LHON
14459 m ND6 LHON or Leighs
14569 m ND6 LHON or Leighs
14484 m ND6 LHON severe
14709 t Glu Varied: infantile
myopathy to NIDDM
14826 cyt b exercise intolerance
15084 m cyt b exercise intolerance
15168 m cyt b exercise intolerance
15257 m cyt b LHON
15498 cyt b exercise intolerance
24nt
del
15723 m cyt b exercise intolerance
15762 m cyt b Myopathy, late-onset
15812 m cyt b LHON
15923 t Thr LIMM Lethal infantile
mitochondrial myopathy
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Position RNA Gene Disease
15990 t Pro MM
16189 D NIDDM Non-insulin dependent
diabetes
N= any nucleotide
R= either puRine (A, G)
Y= either pYrimidine (U, C)
The mitochondrial genome of mitochondria which are in
the cytoplasmic fraction isolated according to the method
of the tenth aspect of the invention can be analysed
according to standard techniques. These techniques are
exemplified further herein and include the polymerase
chain reaction (PCR), restriction fragment length
polymorphism (RFLP) analysis, genomic hybridisation,
nucleotide sequencing and gene function detection and/or
measurement.
In another aspect, the invention provides nucleotides
including the following sequences:
ATP6F: TCACCACCCAACAATGAC
ATP6R: TAAGGCGACAGCGATTTC.
DEFINITIONS:
In the specification and claims, "oocyte" means a
female germ line cell including primary oocytes and
secondary oocytes, and includes human oocytes. Primary
oocytes include germ cells at the GV (germinal vesicle)
stage of meiosis. Secondary oocytes include germ cells at
the metaphase II stage of meiosis.
In the specification and claims, "progeny descended
from a fertilized oocyte" means the individual which is
generated from the fertilization of the female germ cell
with the male germ cell. "Individual" means the
multicellular organism from the earliest stage of
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embryonic life (for example, the 2 cell-stage) to adult
life.
In the specification and claims, "does not impair the
capacity of the oocyte to be fertilized" means that the
oocyte which has had a fraction of cytoplasm isolated, may
be fertilized, or in other words, may undergo any one or
more of the biochemical or cellular events which are
associated with any one or more of the stages of
fertilization, from the activation of the oocyte by entry
of sperm, to the generation of a zygote and the formation
of cleavage products of the zygote. Typically after the
fraction of the cytoplasm is isolated, the oocyte may be
fertilized in vitro by standard techniques, including for
example ICSI and IVF.
In the specification and claims, "embryonic cell"
includes a post-syngamous fusion product of the female and
male germ cells, a zygote, and cleavage products of a
zygote at any stage of development from the 2 cell stage
to the stage of implantation.
In the specification and claims, "progeny descended
from an embryonic cell" or "progeny descended from an
embryo" means the individual which is generated from the
post syngamous fusion product of the female and male germ
cells. "Individual" means the multi-cellular organism from
the earliest stage of embryonic life (for example, the 2
cell stage) to adult life.
In the specification and claims "does not impair the
developmental potential of the cell" or "does not impair
the developmental potential of the embryo" means that the
embryonic cell which has had a fraction of cytoplasm
isolated, and the embryo from which the embryonic cell may
be derived, may undergo any one or more of the biochemical
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or cellular events which are associated-with cell
differentiation and/or maturation.
BRIEF DESCRIPTION OF FIGURES
Figure 1 shows a 400 by fragment amplified from the
D-loop region of the mitochondrial genome derived from
cytoplasmic biopsy samples of oocytes A1 to A7, using
oligonucleotide primers L29 and H04.
Figure 2 shows amplification of a 271 by fragment of
mitochondrial genomes isolated from embryonic cells using
primers corresponding to mtDNA positions 8201 and 8472..
BEST METHOD FOR CARRYING OUT THE INVENTION
1. BIOPSY of OOCYTES
MATERIALS AND METHODS
Source of oocvtes
Human oocytes A1 to A5 were donated for research.
These oocytes were either germinal vesicle (GV) cells or
were at the MI stage of meiosis, and were 6 hours
postretrieval.
Human oocytes B1 to B8 were donated for research.
These oocytes were at the MII stage of meiosis and were 24
hours post-retrieval.
Human oocyte C1 was designated at the patients
request to be part of the study. This oocyte was at the
MII stage of meiosis and was 24 hours post-retrieval.
Isolation of oocyte cytoplasmic fractions
A small amount of ooplasmic material was biopsied
from human oocytes using an ICSI pipette (Sydney IVF,
Sydney). Briefly, the pipette is a glass capillary drawn
out to have an end diameter of approximately 7~m with a
bevelled tip.
The biopsy technique was performed as follows:
ooplasm was drawn into the pipette to a distance of
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approximately 100~tm (approximately 8p1)~. The pipette was
then withdrawn from the oocyte, forming a thin ooplasmic
bridge, which was then broken by stretching. Each
ooplasmic biopsy was expelled directly into a PCR tube
containing 20~..t,lof PCR buffer, Proteinase K and 20 mM DTT.
Tubes were incubated either at 37°C overnight, or at 50°C
for 30 minutes and then frozen. Both protocols were
followed with heat inactivation of Proteinase K at 95°C
for 10 minutes.
Analysis of mitochondrial genome in oocyte cytoplasmic
fractions
The mitochondrial genome in the oocyte cytoplasmic
fractions was analysed by the polymerase chain reaction
( PCR ) .
(i) D-loop fragment
Reactions of 20~.lwere established; 10 ~.l of reaction
mixture was added to 10,1 of ooplasmic biopsy preparation.
The 101 reaction mixture contained 2.5 pmol of each
primer, 200~M of each dNTP, PCR buffer, milli-Q water and
0.5 units of Taq. All reactions were carried out in capped
0.2m1 tube strips. PCR cycling was performed in an FTS
Thermal Sequencer (Corbett Research, Sydney, NSW) under
the following conditions: initial denaturation at 93°C for
5 minutes, followed by 24 to 40 cycles of 93°C
denaturation for 45 seconds, 60°C annealing for 1 minute
and 72°C extension for 1 minute; ending with a polishing
step of 72°C for 7 minutes, cooling to 15°C and holding at
4°C. Complete reaction mixtures lacking template DNA were
included in all PCR reactions as negative controls.
Primers L29 (5'- GGTCTATCACCCTATTAACCAC-3') and H04
(5'-CTGTTAAAAGTGCATACCGCCA-3'), specific for a 400bp
sequence in the mitochondrial D-loop region were used.
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(ii) Common deletion fragment
An identical protocol to that described above was
used, but with the following conditions: initial
denaturation at 95°C for 3 minutes followed by 20 to 35
cycles of 92°C denaturation for 1 minute, 60°C annealing
for 10 seconds and 68°C extension for 45 seconds; ending
with a polishing step of 75°C for 7 minutes, cooling to
15°C and holding at 4°C. From cycle 11, 15 seconds was
added to the extension time every cycle. Complete reaction
mixtures lacking template DNA were included in all PCR
reactions as negative controls.
Primers L820 (5'-TTCATGCCCATCGTCCTAGA-3') and H1363
(5'-GGGGAAGGGAGGTTGACCTG-3'), specific for the 4977 by
common deletion region, require the use of the modified
enzyme, ExpandTM High Fidelity, due to the large fragment
size being amplified and the specialised 'long' PCR
program.
The amplified DNA fragments from the D-loop region or
the common deletion region were analysed by polyacrylamide
gel electrophoresis on a 5% 37:1 acrylamide: bisacrylamide
gel.
Insemination of biopsied oocytes
The B1 to B8 oocytes and the C1 oocyte were
fertilized by either ICSI or IVF according to standard
protocols (1,2). The insemination was performed
immediately after cytoplasmic biopsy of the oocyte.
RESULTS
Oocyte fate after cytoplasmic biopsy
Oocytes A1 to A5 were not visibly affected by the
cytoplasmic biopsy procedure and showed no signs of
degeneration at 48 hours after biopsy. Oocytes B1 to B3
and B5 to B8 showed no signs of degeneration at 48 hours
after biopsy. Oocyte C1 showed no signs of degeneration at
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48 hours after biopsy. A single oocyte,-B4, degenerated at
17 hours after biopsy (Table 2).
Table 2
Oocyte Cytoplasmic 17 hour post 48 hour post
Biopsy biopsy biopsy
A1 YES No degeneration
A2 YES No degeneration
A3 YES No degeneration
A4 YES No degeneration
A5 YES No degeneration
B1 YES No degeneration
B2 YES No degeneration
B3 YES No degeneration
B4 YES Degenerated
B5 YES No degeneration
B6 YES No degeneration
B7 YES No degeneration
B8 YES No degeneration
C1 YES No degeneration
PCR amplification
of mitochondrial
DNA
from
cytoplasmic
biopsy
Co nsideration given to the amplification of
was
mitocho ndria) DNA froma cytoplasmic biopsy taken from
an
oocyte. It was necessary
to calculate
the approximate
number of mitochondriathat would probably be obtained
in
such biopsy to determine
a whether there
would be
suffici ent template
DNA for the
PCR.
Th e cytoplasmic
biopsy was
obtained by
using an ICSI
pipette as described
above. It was
estimated that
the
volume of this cytoplasmic
biopsy would
be approximately
8p1. the volume of
As an oocyte is
approximately
500p1,
the cyt oplasmic biopsyremoved is estimated to comprise
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approximately 2% of the cytoplasm. In a conservative
estimate, there are approximately 100,000 mitochondrial
genomes per oocyte (3). In accordance with this estimate,
the biopsy would remove approximately 1000 mitochondria.
This amount of mitochondria is within the amplification
capabilities of the polymerase chain reaction.
PCR amplification from the D-loop region
A 400 by D-loop fragment was amplified from the
cytoplasmic biopsy of oocytes A1 to A5 with 35 cycles of
amplification (Figure 1).
PCR amplification from the common deletion region
A 5.5kb fragment was amplified from the cytoplasmic
biopsy of oocytes B1 to B3 and B5 to B8 (data not shown).
Insemination of biopsied oocytes
The results of the insemination of the biopsied
oocytes, B1 to B8 and C1 are shown in Table 3.
Table 3
Oocyte IVF/ICSI 48 hour
17 hour post biopsy
post
biopsy
B1 ICSI 2PN 2 cell
B2 ICSI 2PN No division
B3 ICSI 3PN 2 cell
B4 ICSI Degenerated
B5 IVF Not fertilized
B6 IVF Not fertilized
B7 IVF Not fertilized
B8 IVF Not fertilized
C1 ICSI 2PN 2 cell
PN= pronuclei
observed
All oocytes that were subject to the ICSI protocol,
(except B4) fertilized. Two
were pro-nuclei were
observed
in B1, B3 and C1, and 3 pro-nuclei were observed in B3
at
17 hours after
fertilization.
The
oocytes
B1,
B3 and
C1
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divided and progressed to the 2 cell stage at 48 hours
post fertilization. No cell division was observed in B2 at
48 hours.
Although the B5 to B8 oocytes had not degenerated at
48 hours post biopsy, none of these oocytes were
fertilized by insemination via the IVF protocol.
DISCUSSION
Degeneration was observed in only one of the 14
oocytes which were subjected to the cytoplasmic biopsy
technique. As some oocytes tend to degenerate at
approximately 24 hours post retrieval (4,5), it is
possible that the B4 oocyte degenerated independently of
the cytoplasmic biopsy technique. It should be noted that
the oocytes A1 to A5 were matured in a medium formulated
specifically for insemination, rather than oocyte
maturation.
The results show that the cytoplasmic biopsy
technique can be generally applied up to 24 hours post
retrieval of the oocyte. The biopsy technique is therefore
able to be used together with known fertilization
techniques, including for example, ICSI and IVF, which are
generally used approximately 4 to 6 hours post retrieval
of the oocyte. Although in the present study, insemination
was performed immediately after biopsy, it is expected
that the biopsy may be performed after insemination.
We have found that the percentage of oocytes that are
morphologically intact after the ICSI procedure (i.e the
injection of sperm without removal of cytoplasm) is 95.2%
(based on 2649 oocytes injected) (unpublished results).
By way of comparison, the standard for other types of
micro-manipulation was difficult to establish, with the
rate of degeneration after polar body and underlying
cytoplasm removal for nuclear transplantation in other
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species not being documented (6, 7). A survival rate of
30.8% (67/217) quoted for foreign mitochondrial injection
and a 6% lysis rate has been reported with ooplasmic
injection in human oocytes.
Although removal of the cytoplasm is more invasive
than ICSI alone, the overall oocyte survival rate in the
present study is surprisingly higher than our above
discussed survival rates after standard ICSI. Thus the
cytoplasmic biopsy technique can be considered potentially
l0 acceptable clinically.
The amplification of fragments from the D loop region
and the common deletion region of the mitochondrial genome
from mitochondria in the cytoplasmic biopsy demonstrates
that there is a sufficient source of template DNA in the
biopsy sample for analysis by PCR, and that fragments of
the order of from less than 0.5 kb to greater than 5 kb
can be amplified from the sample. The amplification of
fragments from 2 independent loci of the mitochondrial
genome suggests that mutations at other loci of the
mitochondrial genome, in particular the mutations
described in Table 1, can be amplified from the
cytoplasmic biopsy sample using PCR and specific
oligonucleotides.
The fertilization rate and subsequent cleavage rate
was encouraging considering the oocytes donated in these
experimental cohorts were greater than 24 hours old at the
time of cytoplasmic biopsy and attempted fertilization.
Previous studies have indicated that culture for a period
exceeding 20 hours before insemination can compromise
oocyte fertilization and development (4,5). In addition,
oocytes matured in vitro lack the capacity of oocytes
matured in vivo to maintain a high rate of cleavage (8).
For this reason, it was not surprising that the B5 to B8
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oocytes were not fertilized subsequent to insemination via
the IVF protocol.
2. Embryonic cells
Materials and Methods
Source of embryonic cells
Frozen research embryos (3 * two pronuclear embryos,
1 * 2 cell embryo and 1 * 4 cell embryo) were thawed and
equilibrated in growth medium.
Isolation of embryonic cell cytoplasmic fraction
The 3 single cell embryos and 1 cell from each of the
multi cell embryos were biopsied using a standard ICSI
pipette. In each case the pipette was introduced into the
cytoplasmic region and an aliquot of cytoplasm
corresponding to approximately 5% of the cell volume was
withdrawn. This aliquot was delivered into a sucrose
solution. The embryos were returned to the incubator for
continued growth.
Analysis-of mitochondrial genome in embryonic cell
cytoplasmic fraction
The preparations of cytoplasm were made alkaline with
Potassium Hydroxide and heated to 90°C for 5 minutes. After
neutralizing with TrisHCI an aliquot was added to a
reaction mix containing mitochondrial DNA specific primers
designed to amplify a 271 base pair fragment corresponding
to mtDNA positions 8201 and 8472. This region of the
mitochondrial genome comprises part of the coding region
for an oxidase and an ATPase. The mix was PCR amplified
using a thermal cycler capable of real time monitoring of
increasing fluorescence associated with the amplification
of double stranded DNA. Reagent and medium blanks were
negative for any fluorescence changes. Analysis of DNA
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copy number was by comparison to an external dilution of
control human DNA.
RESULTS
PCR amplification of mitochondrial DNA from cytoplasmic
biopsy
All five samples showed the presence of mitochondrial
target DNA. Table 4 shows estimated mtDNA copies in biopsy
samples
Table 4
Embryo type Mt DNA copy number
2PN 2,040
2PN 4,456
2PN 15,304
2 Cell 15,388
4 Cell 10,676
Embryo fate after biopsy
Approximately 24 hours after the biopsy procedure the
embryos were examined using microscopy to ascertain their
individual continued viability. Only 1 of the 2PN embryos
had failed to divide although some degenerating cell
material was observed in the original 2 cell and 4 cell
embryos. Three days later, further observations showed two
embryos had progressed to blastocyst formation.
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REFERENCES:
1. Steptoe, P. and Edwards, R. Pregnancy in an infertile
patient after transfer of an embryo fetrtilized in vitro.
Brit. Med. J. of Clin. Res. 1983; 286: 1351-1352
2. "Intracytoplasmic Sperm Injection, The Revolution in
Male Infertility" Edited by Flaherty, S. P. and Matthews,
C. D. in Reproduction, Fertility and Development, Vol. 7,
No. 2, 1995, CSIRO Australia
3. Chen, X., Prosser, R., Simonetti, S., Sadlock, J.,
Jagiello, G., and Schon, E. A. Rearranged mitochondrial
genomes are present in human oocytes. Am. J. Hum. Genet.
1995; 57: 239-247
4. Trounson, A., O., Mohr, L. R., Wood, C., and Leeton,
J. F. Effect of delayed insemination on in vitro
fertilization, culture and transfer of human embryos. J.
Reprod. Fertil. 1982; 64: 294
5. Plachot, M., de Grouch, J., Junca, A. M., Mandelbaum,
J., Salat-Baroux, J1, and Cohen, J. Chromosomal analysis
of human oocytes and embryos: Does delayed fertilization
increase chromosome imbalance? Hum. Reprod.; 32: 125-127
6. Meng, L., Ely, J. J., Soffer, R. L. and Wof, D. P.
Rhesus monkeys produced by nuclear transfer. Biol Reprod.
1997; 57: 454-459
7. Lavoir, M. C., Kelk, D., Rumph, N., Barnes, F.,
Betteridge, K. J., and King, W. A. Transcription and
translation in bovine nuclear transfer embryos. Bio.
Reprod. 1997; 57: 204-214.
8. Trouson, A., Anderiesz, C., Carson, R., Jones, G.,
MchShane, P., Lolatgis, N., and Wood, C. Oocyte
maturation and growth of human embryos to blastocysts in
vitro. (Abstract) J. Ass. Reprod. Genet. 1997; 14: 472-
473.
