Human Reproduction, Vol. 15, (Suppl. 2), pp. 160-172, 2000
Chromosomal non-disjunction in human oocytes:
is there a mitochondrial connection?
Eric A.Schon1'2'6, Sang Ho Kim1, Jose CFerreira3,
Paulo Magalhaes1, Marcy Grace4, Dorothy Warburton2'5
and Susan J.Gross3
department of Neurology, Columbia University, 2Department of Genetics
and Development, Columbia University, 3Department of Obstetrics and
Gynecology, Montefiore Medical Center/Albert Einstein College of
Medicine, Bronx, NY 10461, 4Armed Forces Radiobiology Research
Institute, Bethesda, MD 20889-5603, and department of Pediatrics,
Columbia University, 630 West 168th Street, New York, NY 10032, USA
6
To whom correspondence should be addressed at: Department of Neurology,
Room 4-431 Columbia University, 630 West 168th Street,
New York, NY 10032, USA.
E-mail: [email protected]
The frequency of chromosome abnormalities due to non-disjunction of maternal chromosomes during meiosis is a function of
age, with a sharp increase in the slope of
the trisomy-age curve between the ages of
30 and 40 years. The basis of this increase,
which is a major cause of birth defects,
is unknown at present. In recent years,
mutations in mitochondrial (mt) DNA have
been associated with a growing number of
disorders, including those associated with
spontaneous deletions of mtDNA (Amt
DNAs). Intriguingly, these pathogenic
AmtDNAs, which are present at extremely
high levels in certain patients, are also
present at extremely low levels (detectable
only by polymerase chain reaction) in normal individuals. The proportion of such
AmtDNAs in normal muscle is a function
of age; the shape of this curve is exponential,
with the accelerating part of the curve
beginning at -30-40 years. We postulate
that, as well as muscle and brain, a similar
160
time-dependent accumulation of AmtDNAs
also occurs in normal oocytes. Since
AmtDNAs are functionally inactive, an
accumulation of such aberrant genomes
could eventually compromise ATP-dependent energy-utilization in these cells. Furthermore, these deficiencies would also
affect the function of the somatic follicular
cells that surround, and secrete important
paracrine factors to, the oocyte. If there is
indeed an age-associated relationship
between AmtDNAs and oocyte age, perhaps
errors in meiosis (which is almost certainly
an energy, and ATP, dependent process) are
related to mutations in mtDNA (primarily
deletions, but perhaps point mutations as
well) in oocytes and/or the surrounding
somatic cells, which result in deficiencies in
both mitochondrial function in general and
oxidative energy metabolism in particular.
This hypothesis would explain many of
the non-Mendelian features associated with
maternal age-related trisomies, e.g. Down's
syndrome.
© European Society of Human Reproduction & Embryology
Oocyte mitochondria and non-disjunction?
Key words: ageing/Down's syndrome/meiosis/
mitochondrial disease/mitochondrial DNA
Introduction
The study of mammalian female meiosis provides us with a fascinating challenge: meiosis
is not only protracted over several years,
but the overall process arrests twice, both in
meiosis I (MI) and meiosis II (Mil). The
primordial germ cells are first seen in fetal
life among the endodermal cells of the yolk
sac at 4 weeks, and following migration to
the gonadal ridge, undergo rapid mitoses,
leading to -7X10 6 oogonia by 20 weeks.
These cells lose their ability to undergo further
mitotic division and subsequently enter meiosis. The exact mechanism that initiates
meiosis is not precisely defined. Ovarian
stromal (pregranulosa) cells surround these
primary oocytes, thus forming the primordial
follicles that will remain in the dictyate stage
of meiosis until maturation of the hypothalamic-pituitary axis at puberty. The majority
of these follicles will undergo various stages
of maturation and atresia, so that at birth only
2X106 germ cells will remain (Peters et al.,
1976). This process of atresia is an ongoing
one, proceeding throughout infancy and
childhood.
With the maturation of the human female
reproductive system, a dominant follicle will
emerge from within a cohort of early stage
follicles. As a result of precise hormonal
control, including autocrine and paracrine signalling, this maturing follicle will continue
through the full spectrum of follicular development. These stages include the primary, preantral, and antral follicular stages, whereupon
the oocyte is surrounded by the somatic
cumulus granulosa cells and the Call-Exner
bodies coalesce into a single antrum containing
the follicular fluid. Following the surge of
luteinizing hormone that presages ovulation,
meiosis resumes, as evidenced by germinal
vesicle breakdown and extrusion of the first
polar body. Meiosis will then proceed until
metaphase in Mil and will remain arrested
until fertilization.
Inhibition and resumption of meiosis
For the purposes of understanding meiotic
control of oocytes, it is helpful to divide
follicular development into two stages, preantral and antral. When removed from a late
stage antral follicle, the oocyte has the capacity
to resume and continue meiosis unhindered to
Mil (Edwards, 1965), a property that preantral
oocytes do not have. Furthermore, co-culture
of the stripped oocytes with granulosa cells
and follicular fluid inhibits meiosis (Tsafriri
and Pomerantz, 1984; Sirard and Bilodeu,
1990). These important observations would
indicate that the inhibition of meiosis in the
preantral oocyte is a result of incomplete
development within the cell itself. Conversely,
the mature antral oocyte is responding to
inhibitory signals from the surrounding
somatic cells and to factors within the follicular fluid. These experiments also demonstrate
that the oocyte must undergo inherent changes
to become competent to resume meiosis. In
murine models, nuclear competence appears
to require both the presence of follicular
somatic cells (Byskov et al., 1997) as well as
the acquisition of the constituents that are
necessary for cell cycle transition. Maturation
promoting factor (MPF) is required for the
resumption of meiosis; it increases in the
oocyte even in the absence of somatic cells
(Eppig et al., 1993). However, nuclear maturation factors do not act in isolation; rather it is
the interplay of extrinsic cell signalling with
intrinsic cell cycle regulators such as MPF
that is critical for the acquisition of nuclear
competence in the oocyte (Chesnel et al.,
1994).
161
E.A.Schon et al.
Age-related changes in maturing
oocytes and follicles
One of the recognized changes in the nuclear
envelope of the mature oocyte is the appearance of microtubule organizing centres
(MTOCs) that will later be used in assembly
of the meiotic spindle. Microtubule motor
proteins are associated with the microtubules
and the chromosomes, particularly the kinetochore (de Pennart et al., 1994) and centrosomes (Battaglia et al., 1996a), and actively
participate in the arrangement and preservation
of the spindle structure. In ageing human
oocytes, irregularities in spindle formation and
chromosome alignment have been identified
(Battaglia et al., 1996b). The actual aetiology
for these changes remains unclear, with a
number of different hypotheses proposed. The
possibility of inherent time-dependency in
the breakdown of the spindle components
themselves has been suggested (Hawley et al.,
1994). However, the astute suggestion has
been made that it could be the necessary
stockpiling of proteins necessary for nuclear
development that is impaired and that ultimately leads to non-disjunction in ageing
oocytes (Hunt and LeMaire-Adkins, 1998). Of
note, changes not just within the oocyte but
in overall follicular functional maturation,
such as decreased time to ovulation, have been
observed in humans (Klein et al., 1996).
Mitochondrial DNA and
mitochondrial disease
The human mitochondrial genome (Figure 1)
is a 16 569 bp circle of double-stranded DNA
(Anderson et al., 1981). It contains genes
encoding two ribosomal RNAs and 22 transfer
RNAs, as well as 13 structural genes that
encode subunits of components of the respiratory chain complexes. Of the 13 structural
genes, seven encode subunits of complex I
(NADH-coenzyme Q oxidoreductase), one
162
Cytb
NDS
ND1
| Sporadic "clonal" deletions (e.g. KSS)
Inherited "multiple" deletions (e.g. AD-fc
ISporadic "multiple" deletions (e.g. ageing)
ND2
ND4
COX I
COX III
COX II
K
A8/6
Figure 1. Map of the human mitochondrial genome (after
Anderson et al., 1981). The genes for the mtDNA-encoded
12S and 16S ribosomal RNAs, the 22 transfer RNAs (1-letter
amino acid nomenclature), and the NADH-coenzyme Q
oxidoreductase (ND), cytochrome c oxidase (COX),
cytochrome b (Cyt b) ATP synthase (A) subunits are all
shown. The origins of light-strand (OL) and heavy-strand
(OH) replication, and of the promoters for initiation of
transcription from the light-strand (LSP) and heavy-strand
(HSP), are shown by arrows (see Clayton, 1982, 2000). The
'common deletion', a AmtDNA species often found in
mtDNA rearrangement disorders and in normal ageing, is
shown, as are point mutations associated with the sporadic
myopathies or the maternally inherited MELAS and MERRF.
(See text for key to abbreviations.)
encodes the cytochrome b subunit of complex
III (CoQ-cytochrome c oxidoreductase), three
encode subunits of complex IV (cytochrome
c oxidase, or COX), and two encode subunits
of complex V (ATP synthetase). Each of these
complexes also contains subunits encoded by
nuclear genes, which are imported from the
cytoplasm and assembled, together with the
mtDNA-encoded subunits, into the respective
holoenzymes, each of which is located in the
mitochondrial inner membrane. Complex II
(succinate dehydrogenase-CoQ oxidoreductase), of which succinate dehydrogenase
(SDH) is a component, is encoded entirely
by nuclear genes; SDH thus serves as an
independent marker for mitochondrial number
and activity.
Oocyte mitochondria and non-disjunction?
Mitochondria (and mtDNAs) are maternally
inherited (Hutchison et al, 1974; Giles et al,
1980). Thus, pathogenic mutations in mtDNA
normally (but, importantly, not always) result
in pedigrees that exhibit maternal inheritance,
i.e. the disease passes only through females,
and essentially all children (both boys and
girls) inherit the error. In addition, because
there are hundreds or even thousands of mitochondria in each cell, with an average of five
mtDNAs per organelle in somatic cells (Satoh
and Kuroiwa, 1991), mutations in mtDNA
typically result in two populations of mtDNAs
(i.e. wild-type and mutated), a condition
known as heteroplasmy.
An ever-growing number of diseases are
due to maternally inherited mtDNA point
mutations (reviewed in Schon et al, 1997).
Point mutations in mtDNA-encoded respiratory chain polypeptides are responsible, in the
main, for Leber's hereditary optic neuropathy
(LHON); neuropathy, ataxia, and retinitis pigmentosa (NARP); and maternally inherited
Leigh syndrome (MILS). The great majority
of mtDNA point mutations, however, are in
the tRNA genes. The most prominent of these
disorders are mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
(MELAS); myoclonus epilepsy with raggedred fibres (MERRF); and a mixed group of
maternally inherited myopathies, encephalomyopathies, and cardiomyopathies (Schon
et al, 1997).
Large-scale rearrangements of mtDNA, i.e.
partial deletions (AmtDNAs) and partial
duplications (dup-mtDNAs), are also associated with disease. The most common pathologies are ocular myopathy (OM) and the KearnsSayre syndrome (KSS), two mitochondrial
disorders associated with progressive external
ophthalmoplegia (PEO) (Holt et al., 1988;
Zeviani et al, 1988; Moraes et al, 1989),
as well as a haematopoietic disorder called
Pearson's marrow/pancreas syndrome (PS)
(Rotig et al, 1989). In these disorders, the
AmtDNAs can be observed easily by Southern
blot hybridization analysis as a large population (up to 80% of total mtDNA) of mtDNA
molecules migrating in electrophoretic gels
more rapidly than full-length mtDNAs.
Importantly, these three syndromes are almost
never maternally inherited, as the deletions
arise spontaneously in the patient whose
mother and siblings are usually normal, both
phenotypically and genetically. In addition,
the deletions are unique in each patient, with
the particular type of deletion differing among
patients; however, about one-third of all KSS/
PEO/PS patients harbour the same deletion,
called the 'common deletion' (Schon et al,
1989; Mita et al, 1990), which removes 4977
bp of mtDNA between the ATPase8 and ND5
genes (see Figure 1). Taken together, these
findings imply that the AmtDNA population
in any one such patient is a clonal expansion of
a single spontaneous deletion event occurring
early in oogenesis or in embryogenesis.
Some mtDNA rearrangements are maternally inherited, but these patients do not have
the clinical features of KSS or PEO; they
more typically have maternally inherited type
II diabetes (Dunbar et al, 1993). Significantly,
mtDNA duplications, but not mtDNA deletions, have been documented to be inherited in
this way. With but one exception (S.DiMauro,
personal communication), there is no convincing evidence that large-scale mtDNA deletions can be inherited through the female
germline, implying that high levels of
AmtDNAs (but, somewhat surprisingly, not
equivalently high levels of pathogenic mtDNA
point mutations) are incapable of being transmitted to viable offspring.
Besides the 'clonal' mtDNA deletions found
in the sporadic rearrangement disorders, a
number of Mendelian-inherited disorders in
which affected family members harbour large
quantities of multiple species of deletions of
mtDNA in tissues (usually muscle) have been
described. These deletions are apparently gen163
E.A.Schon et al.
erated during the lifespan of the patient
(Zeviani et al, 1989; Nishino et al, 1999).
Intriguingly, while most mtDNA point
mutations can be maternally transmitted (and
maternally inherited), a growing group of
mutations appear to violate this dictum. Of
the few known sporadic mtDNA point
mutations or microdeletions (-10% of all
point mutations), almost all are located in
polypeptide-coding genes. While they are predominantly in the cytochrome b subunit of
complex III (Dumoulin et al, 1996; Andreu
et al, 1998, 1999a), these mutations have also
been found in other complexes (Keightley
et al, 1996; Hanna et al, 1998; Andreu et al,
1999b). The prevalence of sporadic mutations
in mtDNA-encoded polypeptides implies that,
like AmtDNAs, the loss of a functional respiratory chain somehow selects against viable
progeny in the germline.
Deletions of mtDNA in somatic tissues
during normal ageing
In the last few years it has become clear that
tissues from normal individuals, especially
terminally differentiated (post-mitotic) tissues
with high oxidative requirements, e.g. muscle
and brain, contain low amounts of AmtDNAs
(Schon et al, 1996). These deleted mtDNAs,
which are observable only after amplification
using the polymerase chain reaction (PCR), are
qualitatively identical to the highly abundant
populations of AmtDNAs that have been
described in patients with sporadic PEO and
KSS, sporadic Pearson's syndrome, and
autosomal-dominant PEO.
Importantly, the AmtDNAs found in normal
individuals appear to accumulate during ageing. Using a quantitative PCR technique to
measure the amount of the common deletion,
we have found that this species of AmtDNA
accumulates in muscle by a factor of 10 000
over the course of the normal human lifespan,
reaching a level of -0.1% of total muscle
164
mtDNA by the age of 84 years (Simonetti
et al, 1992; Pallotti et al, 1996). Besides
the common deletion, numerous other deleted
species are also present in ageing muscle
(Zhang etal, 1992; Chen et al, 1993; Pallotti
et al, 1996). Thus, both the overall number
of different AmtDNA species, and the amount
of each such species, increases with time, such
that, in the aggregate, perhaps as much as 510% (or, according to some workers, even
more) of total mtDNA is deleted in elderly
individuals. These AmtDNAs are not distributed homogeneously, at least in muscle.
Rather, individual muscle fibres appear to
contain the majority of deleted molecules
(Schon et al, 1996; Brierley et al, 1998), and
it is these fibres that are respiratorily deficient.
Presence of mtDNA deletions in
normal human oocytes
We currently do not know why, or how,
AmtDNAs accumulate with age. A logarithmic
increase in AmtDNAs with respect to age
would be consistent with the idea that
AmtDNAs are competent for replication, and
the AmtDNAs observed at later ages are progeny of AmtDNAs that arose earlier in time.
This exponential accumulation of AmtDNAs
poses an interesting mechanistic question.
AmtDNAs appear to arise afresh with each
generation, as our data indicate that there are
four orders of magnitude fewer AmtDNAs in
infancy as compared to old age. If this is true,
how can a woman of childbearing age, who
already harbours a low but detectable level of
AmtDNAs (at least in her somatic tissues),
give birth to a baby containing almost no
AmtDNAs at all? In other words, how does
the organism 'reset' the level of mtDNA
mutations in each generation? This may be
due to one of at least three possibilities: (i)
AmtDNAs arise and accumulate in somatic
tissue, but do not arise in female germline
tissue (a hypothesis proposed by Nagley et al,
Oocyte mitochondria and non-disjunction?
1992); (ii) AmtDNAs do arise in oocytes, but
are actively eliminated via some unknown
mechanism; or (iii) AmtDNAs do arise prior
to or during oogenesis but are rarely transmitted to the viable embryo because they cannot
pass through the narrow mitochondrial population bottleneck during oogenesis and subsequent embryogenesis (Hauswirth and Laipis,
1985; Ashley et al, 1989; Jenuth et al, 1996,
1997). The massive accumulation of a single
species of AmtDNA in spontaneous KearnsSayre syndrome (in which the population of
deleted molecules is present in all tissues, and
is presumably a clonal expansion of an initial
deletion event occurring early in oogenesis or
embryogenesis) is consistent with all three
possibilities, but is perhaps most compatible
with the last one.
In 1995, we began to address this question.
Using single-oocyte PCR, we were able to
quantify the total amount of mtDNA in unfertilized human oocytes left over from an IVF
programme: the average oocyte contained
-100 000 mitochondrial genomes (Chen et al,
1995). In addition, we detected a single marker
AmtDNA4977 (the 4977 bp common deletion)
in about half of all the oocytes we tested. The
amount of this species of AmtDNA varied
among oocytes, but was 10-100 such molecules per oocyte (Chen et al., 1995). These
results have been confirmed by some groups
(Keefe et al, 1995; Brenner et al, 1998), but
not by others (Miiller-Hocker et al, 1996).
Deletions of mtDNA have also been observed
in ageing ovarian tissue (Kitagawa et al, 1993;
Suganuma et al, 1993) and in spermatozoa
(Reynier et al, 1998).
Hypothesis
Maternal meiotic non-disjunction resulting in
aneuploidy shows a very strong relationship
with increasing maternal age, with an exponentially rising curve after -32 years. This
maternal age effect holds for all chromosomal
pairs, with the exception of the very large
chromosomes (where there seems to be little
maternal age effect) and for chromosomes 2
and 16 (where the effect is linear) (Warburton
and Kinney, 1996). It has been estimated that
at least 30% of oocytes in women aged >40
years have undergone non-disjunction.
These findings have stimulated us and others
(Jansen and De Boer, 1998; Van Blerkom
et al, 1998) to consider the possibility that
an age-related increase in mtDNA mutations
in oocytes could be an underlying cause of
the maternal age effect on chromosomal nondisjunctions. Specifically, we postulate that,
as a woman ages, mtDNA mutations accumulate in her oocytes, and that the frequency
of aneuploidy increases once a threshold of
mitochondrial energy deficit is crossed. As
described above, the process of meiotic inhibition and resumption is the result of the production of autonomous factors by the oocyte, as
well as the response to extrinsic signalling by
the surrounding follicular cells. Consequently,
mtDNA mutations that accumulate over time
in the surrounding somatic cells might also
lead to aneuploidy. This 'mitochondrial ageing
hypothesis' would explain the relationship
between maternal age and the frequency of
trisomies, e.g. Down's syndrome.
The idea that oocytes require high concentrations of ATP (Van Blerkom and Runner,
1984; Van Blerkom, 1991) and that a mitochondrial energy deficit could result in aneuploidies is not a new one (Beermann and
Hansmann, 1986). However, the most compelling reason to consider a mitochondrial aetiology in germline aneuploidies may be that
there are no mechanistic data that explain
successfully the age-related increase in human
trisomies. Specifically, no data exist to explain
why the mere passage of time should result
in the increased frequency, of non-disjunction
events. An age-related phenomenon can be
the result, of course, of age-related changes
in nuclear DNA, or to epigenetic and environ165
E.A.Schon et al.
mental factors, but it is more difficult to
invoke these causes (which probably vary
from individual to individual) to explain the
consistent and specific observation of agerelated aneuploidies, which show little or no
variation with geographical region, ethnicity,
or social class. On the other hand, the 'mitochondrial paradigm', with its focus on population genetics, on random but cumulative
effects of mutations, on heteroplasmy (and, in
ageing, 'polyplasmy'), and on mitotic segregation (coupled with the mitochondrion's
monopoly in oxidative energy metabolism) is
an attractive alternative hypothesis to explain
the ageing-aneuploidy correlation.
Other facts also support a mitochondrial
aetiology. As noted above, there is an exponential increase in the amount of the common
deletion in muscle over time (Simonetti et al,
1992; Pallotti et al, 1996). The shape of this
curve roughly parallels the curve describing
the increase in the frequency of trisomies as
a function of maternal age (Penrose, 1933;
Hassold and Jacobs, 1984; Hassold and Chiu,
1985), that is, the curve begins to accelerate
at -30-40 years.
The vast majority of all chromosomal nondisjunction events are maternal, not paternal
(although for a few specific chromosomes,
e.g. chromosome 2, paternal disomies may
predominate) (Zaragoza et al, 1998), and
nearly 80% of maternal non-disjunctions occur
during meiosis I (Antonarakis et al., 1991,
1992, 1993; Sherman et al., 1991; McFadden
etal., 1993; Abruzzo and Hassold, 1995). The
frequency of Mil disomies in comparison
with MI disomies varies from chromosome to
chromosome (Fisher et al., 1995; Hassold
et al., 1995), but it appears that both MI and
Mil errors increase with advancing maternal
age (Yoon et al., 1996). However, there are
also data to suggest that all non-disjunction
events are the result of errors in MI (Lamb
et al., 1996). In either case, we believe it is
significant that oocytes are generated only
166
during fetal life and have the same age as the
woman bearing them.
Data from other organisms such as yeast and
Drosophila have shown that factors causing
decreased recombination result in an increase
in meiotic non-disjunction and aneuploidy.
Analysis of recombination events in human
trisomic conceptions has demonstrated a consistent decrease in recombination for maternal
non-disjunctive events classified as meiosis I,
as well as a change in the chromosomal
distribution of these recombination events
(Lamb et al, 1996, 1997; Robinson et al,
1998). For Mil errors, the results have been
less consistent across chromosomes (Bugge
et al, 1998), but for trisomy 21, an increase
in recombination in pericentromeric regions
was described.
These data have led to the hypothesis that
the maternal age effect on non-disjunction is
a 'two-hit' process. In the first hit, the underlying distribution of recombination events per
chromosome bivalent, established during fetal
life, creates pairs susceptible to non-disjunction, either because of too little or too much
pericentromeric recombination. While a young
ovary can handle most configurations successfully, older oocytes are likely to undergo
non-disjunction, given these less than optimal
recombination patterns (Lamb et al, 1996).
In the second hit, our hypothesis concerning
the role of accumulating mitochondrial DNA
mutations addresses the reason for the change
in oocyte competence with maternal age.
Chromosomal disjunction, whether in meiosis I or meiosis II, is almost certainly a highly
energy-intensive and ATP-dependent process,
because the synapsed chromosome pairs (in
MI) or sister chromatids (in Mil) must be
pulled to the opposite ends of the dividing
oocyte along the 'pulley-and-rope' machinery
of the mitotic spindle apparatus (EichenlaubRitter et al, 1996; Eichenlaub-Ritter, 1998).
Recent studies have shown that, in vitro,
matured oocytes taken from antral follicles of
Oocyte mitochondria and non-disjunction?
ageing women show spindle abnormalities
and problems with chromosome alignment
(Battaglia et al, 1996a; Volarcik et al, 1998).
Moreover, it has been shown that an increased
frequency of oocyte aneuploidies in mice can
be related to a specific mitochondrial genotype
in the ooplasm (Beermann et al, 1988).
We speculate that mitochondria and
mtDNAs in oocytes turn over in order to
provide a certain minimum level of energy for
oocyte viability. The supposition that mtDNA
replicates in the resting oocytes of primordial
follicles (currently unverified experimentally)
would provide a means by which mutations
in mtDNA could arise in otherwise quiescent
oocytes and then accumulate over time. Likewise, the surrounding somatic cells, including
granulosa precursors, must maintain viability
over many years.
The accumulation of mtDNA mutations
could have harmful effects on mitochondrial
energy production and on overall energy levels
in the oocyte. There is only one mitochondrial
genome per organelle in oocytes (Michaels
et al, 1982; Piko and Taylor, 1987), as
opposed to an average of five mtDNAs per
organelle in somatic cells (Satoh and Kuroiwa,
1991). Thus, oocytes should be particularly
prone to the effects of mtDNA mutations, as
mutation in only a single mtDNA molecule
could theoretically impair the function of the
entire organelle. We also note that the mitochondrial respiratory chain is the single
greatest source of free radicals in the body,
and that cumulative defects in mtDNA integrity could be intimately associated with damage by reactive oxygen species. Thus, the
accumulation of mtDNA mutations might not
only affect ATP production directly, but could
impair it indirectly and/or secondarily, through
the downstream effects of free radicals and
oxidative stress on cellular functions (Tarfn,
1996; Tarin et al, 1996; 1998).
Regarding the oocyte-follicle as a single
functional unit may also aid in our understand-
ing of mitochondrial perturbations, in two
ways. First, endocrinopathies are a major feature of mitochondrial disorders. The ovarian
follicle would certainly fit the description of
an active and dynamic endocrinological unit,
particularly during maturation, which is under
tightly regulated, sequential hormonal control,
with secretion of sex steroids as well as growth
factors and regulatory peptides. Second, the
follicular fluid contains a plethora of molecules
secreted by the somatic cells, including mitochondrion-derived steroids. Granulosa development, in fact, is accompanied by increases
in the numbers of mitochondria and topographical shifts in their subcellular location (Meidan
et al, 1990; Dive et al, 1992). This supposition is consistent with the suggestion that
mitochondrial mutations are particularly prone
to disrupt molecular transport across cell membranes (Schon et al, 1997). Thus, mitochondrial mutations could theoretically alter
membrane transport and thereby alter the signals perceived by the oocyte.
Problems with the hypothesis
There are at least two key pieces of evidence
that do not support this hypothesis. First,
there is no evidence that levels of pathogenic
mtDNA mutations high enough to cause overt
disease can cause karyotypic abnormalities in
patient cells. For example, we were unable to
detect karyotypic changes in fibroblasts from
MERRF patients harbouring >80% mutated
mtDNAs (mutation in the tRNALys ^ene), even
though it is known that high levels of the
MERRF mutation can impair mitochondrial
protein synthesis (Masucci et al, 1995). This
result implies that either an extremely high
level of mutant mtDNAs are required before
mitotic non-disjunction occurs (i.e. that nondisjunction is a threshold phenomenon), or
that chromosomes can divide well even with
reduced ATP values. Of course, evidence
167
E.A.Schon et al.
obtained on mitotic cells may not be applicable
to meiotic cells.
A potentially more serious problem for the
hypothesis is the fact that women harbouring
high levels of pathogenic mtDNA point
mutations (usually 50-70% of total mtDNA)
are able to have children who are destined to
have a serious mitochondrial disorder, yet
whose cells are karyotypically normal. This
implies that chromosomes in oocytes with
severely reduced levels of ATP can still replicate and divide normally. On the other hand,
the failure to transmit mtDNA deletions from
mother to child, and the extremely high prevalence of spontaneous cytochrome b mutations
(and the lack of reported maternally inherited
cytochrome b mutations) implies that at least
some types of mtDNA mutations interfere
with germline or zygotic viability. Examining
reproductive histories of women bearing children with mitochondrial disorders for
increases in infertility, in spontaneous abortions, or in trisomic offspring might be worthwhile in this context.
Predictions of the hypothesis
The hypothesis that mtDNA mutations are
associated with trisomies predicts a number
of consequences, all of which can be tested
experimentally.
First, it predicts that the proportion of
mtDNA mutations in oocytes increases as a
function of age, irrespective and independent
of the presence of aneuploidies in individual
germ cells. This issue can be addressed by
quantifying mtDNA deletions in oocytes versus age, using methods already described
(Chen et al, 1995).
Second, it predicts that the proportion of
mtDNA mutations is higher in oocytes harbouring aneuploidies than in normal cells,
irrespective of age. This issue can be addressed
by performing simultaneous karyotyping and
quantitative mtDNA analysis on individual
168
oocytes. In addition, if non-disjunction is due
to a high proportion of mtDNA mutations in
the oocyte itself, and if this high level of
mutation is transmitted to the fetus (i.e. no
loss through the bottleneck in early embryogenesis), the hypothesis predicts that the
proportion of mtDNA mutations ought to be
greater in the somatic cells of trisomic patients
or fetuses than in normals, and that patients
with trisomies might have mitochondrial diseases at a relatively high frequency.
Third, it implies that mtDNA mutations in
oocytes should have detectable biochemical
consequences, such as loss of cytochrome c
oxidase activity (but see Mtiller-Hocker et al.,
1996). This can be tested by performing respiratory chain histochemistry and immunohistochemistry (Bonilla et al., 1992) on karyotyped
oocytes.
Fourth, it also implies that there is mtDNA
turnover (presumably by replication) in germline tissue. This might be able to be verified
experimentally via pulse labelling of mtDNAs
in vivo (Moraes and Schon, 1995).
Finally, if aneuploidy is primarily the result
of energy deficits in the follicular cells, there
ought to be a correlation between the level of
mtDNA mutations in granulosa cells and the
presence of aneuploidy in the oocyte contained
within that follicle. This can be tested, for
example, by performing in-situ hybridization
on sectioned follicles to detect AmtDNAs
(Mita et al, 1989).
Positive results from experiments of these
types would not only support a mitochondrial
aetiology for age-related aneuploidies, but
might also point the way towards rational
approaches to prevention of these devastating
birth defects.
Acknowledgements
This work was supported by grants from the National
Institutes of Health (NS28828, NS11766, and HD32062)
and the Muscular Dystrophy Association.
Oocyte mitochondria and non-disjunction?
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