Human Reproduction, Vol. 15, (Suppl. 2), pp. 92-101, 2000
Fertilization and elimination of the paternal
mitochondrial genome
J.M.Cummins1
Division of Veterinary and Biomedical Sciences, Murdoch University,
Western Australia 6150
'To whom correspondence should be addressed at: Division of Veterinary and
Biomedical Sciences, Murdoch University, Western Australia 6150.
E-mail: [email protected]
With rare exceptions, mammalian mitochondria are inherited through the female.
This probably serves to minimize lethal
cytoplasmic gene competition and to prevent the inheritance of sperm mitochondrial
DNA that has been subject to degradation
by free radicals. In general, organisms are
intolerant of mitochondrial heteroplasmy
and, when this occurs in humans, it frequently presents as progressive and lethal
bioenergetic or neurological disease. The
mitochondria of spermatozoa are specifically destroyed by proteolysis in early
embryonic development, in mice at the 4to 8-cell transition. While there are concerns
in human assisted reproduction that microinjection of abnormal or immature sperm
cells could lead to lasting harm in the
offspring through transmission of abnormal
mitochondria, there is no clinical evidence
to support this. There is more potential for
harm through attempts to 'rescue' poor
quality oocytes by cytoplasmic or nuclear
transfer, as it is not currently possible to
control the final fate of the donated mitochondria in relation to nuclear-mitochondrial interactions or the embryonic axes.
Moreover, the balance between nuclear and
mitochondrial genes and the role of cytoplasmic factors in epigenesis are still poorly
92
understood. The future challenge for biologists is to comprehend the nature of the
selective destruction of paternal mitochondria, as it appears to be a species-specific
recognition phenomenon.
Key words: embryogenesis/fertilization/maternal inheritance/mitochondrial DNA/mitochondrial transfer
Introduction
This review will attempt to summarize the
latest knowledge on the nature of mammalian
mitochondrial inheritance and its implications
for human assisted reproductive technology.
There are comprehensive accounts of the place
of the mitochondria in the life cycle (Smith
and Alcivar, 1993; Ozawa, 1997; Cummins,
1998a). Epigenesis and genomic imprinting in
relation to mitochondrial inheritance will be
only briefly touched upon; the interested reader
should consult recent reviews (John and
Surani, 1996; Latham, 1999).
Mitochondrial inheritance: an overview
Mitochondria exist as a set of semi-autonomous organelles with their own restricted genome of 16.6 kb in humans (much larger
© European Society of Human Reproduction & Embryology
Fate of paternal mitochondria in embryos
genomes are found in plants and invertebrates). Ancestrally, mitochondria probably
derived from endosymbiotic purple bacteria
as a means of exploiting oxygen (Margulis
and Sagan, 1986; Gray, 1992; Taanman, 1999).
In contemporary eukaryotes, mitochondria
have sacrificed much of their genome to the
somatic cell nucleus and serve as 'slave'
organelles, carrying out the risky business of
oxidative phosphorylation well away from the
free-radical-sensitive 'master' nuclear genome. Approximately 200 control genes are
found in the nucleus, compared with only
37 in the highly compressed genome of the
mitochondrion (Shadel and Clayton, 1997;
Taanman, 1999). Control is therefore complicated, involving much exchange of information
between the nucleus and the many copies
of mitochondrial (mt) DNA in each cell's
cytoplasm (Poyton and McEwen, 1996; Shadel
and Clayton, 1997). With very rare exceptions,
mitochondria and other cytoplasmic factors
(e.g. centrioles) are inherited uniparentally in
eukaryotic organisms (Birky, 1995; Simerly
et al, 1995; Hewitson et al, 1998). For
mitochondria, transmission is normally
through the female germ cell line, but there are
examples of paternal inheritance (for example
some conifers in which the male gamete forms
a pollen tube). There are some unusual modes
of mixed inheritance, for example in mussels,
in which paternally derived mitochondria are
transmitted to male offspring, but maternal
mitochondria pass to both genders (Zouros
et al, 1992). However, these seem to be
exceptions to the general rule. The mechanisms that prevent dual parental inheritance are
extremely diverse and have probably evolved
separately many times (Birky, 1995). The
evolutionary reasons for this are obscure. It
has been suggested (Hurst, 1992; 1995) that
it may serve to avoid lethal genome conflict
among subservient yet essential organelles. An
alternative and perhaps more readily testable
hypothesis is that the spermatozoon's mtDNA
is damaged by reactive oxygen species (ROS)
during its life and would thus be detrimental
to the embryo if allowed to contribute to the
zygote (Cummins et al, 1994; Allen, 1996;
Cummins, 1998a). The asymmetric life history
between maternally and paternally derived
mitochondrial DNA could even be central
to the evolution of two genders with their
complementary life histories (Short, 1998).
The ancestral experimentation that resulted in
the present combination of meiosis, amphimixis and amphogeny among contemporary
eukaryotes, thus preceded later developments
such as the evolution of genomic imprinting
in eutherian mammals (Bell, 1982; Margulis
and Sagan, 1986; Latham, 1999).
A healthy complement of founding mitochondria is essential for embryonic growth
and fitness. This is apparently accomplished
by a stringent genetic bottleneck in early
embryogenesis and is possibly reinforced by
the selective elimination of oogonia with
defective mtDNA (Cummins, 1998a; Jansen
and de Boer, 1998). Various lines of evidence
indicate that mitochondria in oogonia and
the early embryo are quiescent and hence
relatively unlikely to engender damaging ROS
(Allen, 1996). Indeed replication of mtDNA
in the mouse embryo does not start until
the egg cylinder stage at day 6.5 (Piko and
Matsumoto, 1976; Piko and Taylor, 1987;
Taylor and Piko, 1995). One report suggests
that all mtDNA in the mature human may
effectively derive by clonal expansion from a
single precursor copy (Blok et al., 1997), but
others report higher source numbers (Jenuth
et al., 1996), The bottleneck, coupled with
stringent selection, also serves to counteract
Muller's ratchet: the inexorable accumulation
of deleterious mutations in asexually reproducing organisms (Bergstrom and Pritchard,
1998). The evidence linking oocyte atresia
with specific mtDNA defects, as opposed to
simple ageing, is scarce (Chen et al., 1994;
Keefe et al, 1995; Brenner et al, 1998).
93
J.M.Cummins
Nevertheless this is an area that deserves more
research as it may give a key to the diminishing
fertility of the mammalian oocyte with age
(Adams, 1984; Jansen and de Boer, 1998).
Many aspects of mitochondrial function that
are 'standard' in undergraduate texts may now
be over-simplifications or simply untrue. For
example, the common assertion that mitochondria lack mechanisms to repair oxidative damage to DNA appears to be false, as they
possess efficient base and nucleotide excision
repair pathways (Bohr et al, 1998; Bohr and
Dianov, 1999). It is also commonly stated
that mtDNA mutates much more rapidly than
nuclear DNA. This is true as a statement of
averages, but the mutation rate is actually
highly variable across the mitochondrial genome. Some regions show nucleotide substitution rates similar to nuclear DNA, whereas
synonymous sites and small rRNAs mutate
-20 times more rapidly and tRNAs -100 times
more rapidly than in their nuclear counterparts
(Pesole et al, 1999). The doctrine that mtDNA
does not show recombination is also shaky.
Mitochondria possess the requisite enzyme
systems in vitro (Thyagarajan et al, 1996)
and two very recent independent studies have
documented recombination in human populations (Eyre-Walker et al., 1999; Hagelberg
et al., 1999). As mtDNA is normally sequestered inside a relatively impermeable double
capsule, it is difficult to see how recombination
could occur without fusion of paternal and
maternal organelles early in development (see
below). These observations have yet to be
absorbed by molecular biologists attempting
to reconstruct ancestral human populations
based on mtDNA (and Y chromosome)
sequences (Seielstad et al., 1998). Assumptions about the steadfastness of the mtDNA
and Y chromosome 'molecular clocks' are
evidently under challenge.
Sperm mitochondrial fate after
fertilization
In the majority of mammals, the entire sperm
tail enters the oocyte at fertilization. Although
94
this has been known since the last century, it
has been overlooked by the present generation
of molecular biologists, intent on validating
the 'African Eve' theory of human origins
based on exclusive maternal inheritance of
mtDNA (Ankel-Simons and Cummins, 1996).
However, except in unusual cases such as
inter-species crosses (Gyllensten et al., 1985,
1991; Kaneda et al, 1995; Shitara et al,
1998), the mitochondria apparently do not
normally survive.
In most embryos, the sperm midpiece can
be seen to degenerate within 1-2 days of
fertilization (Cummins, 1998a). This is not
merely a temporal decay. In mouse embryos
injected with spermatozoa bearing fluorescently labelled mitochondria, intact midpieces
have been observed up to the 4-cell stage (day
2) but no later. In some cases, however,
mitochondria have persisted for up to 5 days,
but only in embryos that had their development
arrested at the 4-cell stage or earlier (Cummins
et al, 1997). This suggests that disappearance
of the mitochondria might be keyed in to
critical phases of the second mitotic division,
as observed in other mammals (including
humans) that have been studied (Cummins,
1998a). Similar results have been obtained
using spermatids (Cummins et al, 1998b)
and spermatocyte cytoplasm (Cummins et al,
1999), suggesting that whatever destines
sperm mitochondria for destruction in the
embryo is established by the time cells are
committed to spermatogenesis. Kaneda et al.
concluded that destruction of the sperm's mitochondrion probably depends on nuclearencoded factors and not on recognition of the
mtDNA itself; this was because the paternal
mtDNA could be detected in inter-specific
crosses of Mus musculus with M.spretus, but
could not be detected in crosses oiM.musculus
with the congenic strain b6.mt(spr), which
carries M.spretus mtDNA on a background of
M.musculus nuclear genes (Kaneda et al,
1995). The same group found that paternally
Fate of paternal mitochondria in embryos
derived mtDNA in interspecies crosses does
not persist beyond the Fj generation (Shitara
et al., 1998), but this has yet to be confirmed
by independent investigators.
There are several possible reasons for the
lack of persistence of paternal mtDNA (AnkelSimons and Cummins, 1996; Cummins,
1998a). One is that simple dilution of the 75
or so sperm mitochondrial copies among the
100 000 (or more) of the oocyte makes it very
difficult to detect the paternal contribution by
current molecular techniques such as PCR,
which tends to amplify the dominant DNA
sequence selectively in a mixed sample.
Alternatively, the sperm mtDNA could be
simply degraded by excessive exposure to
ROS (Allen, 1996; Aitken et al, 1998) or
unequally sequestered to a region of the forming embryo that does not contribute to the
inner cell mass (Birky, 1983, 1995). Sperm
mitochondria seem inherently dysfunctional
as, unlike somatic cell mitochondria, they
cannot colonize mitochondrially depleted cell
lines (King and Attardi, 1989).
The most convincing evidence for an active
as opposed to a passive mechanism of sperm
midpiece destruction was reported by Sutovsky (from the Oregon Regional Primate
Center, USA) at the 38th Annual Meeting of
the American Society for Cell Biology in
San Francisco in 1998 (cited by Hopkin,
1999). Sutovsky had previously suggested that
spermatozoa could be tagged with ubiquitin
and thus targeted for degradation in the zygote
by 26S proteasome (Sutovsky et al, 1996,
1997b; Sutovsky and Schatten, 1997). This is
an attractive hypothesis, as ubiquitin-mediated
degradation is seen in a wide range of cell
regulation processes, including the recycling
of cell cycle components, class I antigen
turnover, receptor-mediated endocytosis, and
signal transduction pathways (Hochstrasser,
1996). Fluorescent antibodies to ubiquitin
were observed on the developing sperm midpiece in the testis. Although mature spermato-
zoa do not fluoresce, the signal reappears after
the spermatozoon enters the oocyte. Sutovsky
therefore postulates that the polypeptidelinked ubiquitin is masked on the spermatozoa
by disulphide bonds during epididymal maturation and that these are reduced by high
levels of glutathione after oocyte penetration
(P.Sutovsky, personal communication). While
this intriguing finding needs independent confirmation, it seems a very plausible mechanism. As ubiquitin is likely to be present in the
early mitotic phase of spermatogenesis (for
cell cycle component turnover), it would entail
a simple set of selective steps to enable it to
persist on the midpiece and thus ensure later
destruction by the embryo. It is still not known
what aspect of the mitochondrial sheath would
be targeted, but the sperm mitochondria are
significantly modified during spermatogenesis
and develop some unique haploid-encoded
products, including a cysteine-rich selenoprotein capsule protein (Cataldo et al., 1996).
The question remains of how recombination
events can (rarely) occur between sperm and
oocyte mitochondria. One possibility suggested by Sutovsky (personal communication)
is that soon after fertilization a few mitochondria are displaced from the neck region to
allow contact between the sperm centriole and
the oocyte cytoplasm (Sutovsky et al., 1996,
1997a,b; Sutovsky and Schatten, 1997). In
most mammals (but not the murid rodents),
microtubule formation catalysed by the sperm
centriole serves to bring the male and female
pronuclei into apposition and to form the first
cleavage spindle apparatus (Simerly et al.,
1996; Hewitson et al., 1997). One or more
of these early-departing mitochondria might
evade proteolysis by fusing with an oocyte
mitochondrion to establish a heteroplasmic
founder line. Survival would be most unlikely,
as the overall contribution of mtDNA to the
zygote would be <1 in 100 000 (AnkelSimons and Cummins, 1996). However this
is approximately the same order of magnitude
95
J.M.Cummins
Table I. Outcome of mitochondrial transfer to mouse zygotes
Mitochondrial source
Technique
Tracking
method
Survival of
mitochondria
Reference
Testis, liver
Liver, different strain
Spermatocytes
Spermatids
Spermatozoa
Human MELAS patient
Mouse zygotes
Microinjection
Microinjection
Microinjection
Microinjection
Microinjection
Microinjection
Karyoplast/
cytoplast fusion
PCR
PCR
PCR
MitoTracker®
MitoTracker®
PCR
PCR
No
Yes
No
No
No
Yes
Yes, but
variable
results
Ebert et al. (1989)
Pinkert et al. (1997)
Cummins et al. (1999)
Cummins et al. (1998b)
Cummins et al. (1997)
Rinaudo et al. (1999)
Jenuth et al. (1996 (1997)
Meirelles and Smith (1997 (1998)
Van Blerkom et al. (1998)
PCR = polymerase chain reaction; MELAS = mitochondrial encephalomyopathy with lactic acidosis and stroke-like events.
as the paternal 'leakage' of mtDNA observed
in repeated back-crosses between M.spretus
with M.musculus (Gyllensten et al., 1985,
1991). Indeed one has to question whether
these original results in fact reflect such leakage and are not simply the establishment of a
stable heteroplasmic line through a recombinational event. Mitochondrial fusion and fission
are normal in oogenesis and embryogenesis
(Smith and Alcivar, 1993), so rare fusion
events involving sperm mitochondria may be
possible.
Experimental mitochondrial transfer
Mitochondrial inheritance has now been
studied in a variety of cells and tissues. The
results of transfer from various sources to
embryos are summarized in Table I. It is
clear that mitochondria from all stages of
spermatogenesis are unique in failing to persist
following transfer. The fate of transferred or
fused somatic and zygotic mitochondria is
highly variable. This is almost certainly due
to fortuitous placing of the mitochondria with
relation to the nucleus and to the developing
embryonic axes. Mitochondrial replication in
cells starts in cytoplasmic regions closest to
the nucleus, probably as a result of diffusion
or transport of critical control factors (Shadel
and Clayton, 1997). It is also now recognized
that, like the non-mammalian vertebrates,
96
mammalian oocytes and embryos have welldefined axes: these determine the planes of
cleavage and the fate of blastomeres destined
to form the inner cell mass, as distinct from
the trophectoderm (Edwards and Beard, 1997;
Antczak and Van Blerkom, 1997, 1999;
Van Blerkom, 1998).
The presence of embryonic axes and tissue
commitment at such an early stage of development raises a central question about the inheritance of mitochondria, as the next generation
can only derive from the germ cell lineage
(Weissman, 1891). In Drosophila, the gametes
derive from early 'pole cells' formed in the
embryo. In Caenorhabditis elegans and in
zebra-fish, asymmetric segregation of cytoplasm during consecutive cell divisions eventually results in a distinct germ cell lineage.
In mice and presumably in all mammals, germ
cells appear to be delineated relatively late in
development, midway through gastrulation, at
-7.25 days after fertilization (thus <1 day
after the beginning of mtDNA replication);
they can be detected as large, alkaline-phosphatase cells in the yolk sac close to the
forming allantois (Byskov, 1982). McLaren
suggests that germ cell status might be the
result of differentiation in response to local
signals, possibly from somatic tissues such as
the trophectoderm and primitive endoderm
(McLaren, 1999). In this case, germ cell
formation, and hence the transmission of mito-
Fate of paternal mitochondria in embryos
chondria to the next generation, could be more
a matter of timing than of positioning. This is
a highly active area of research with considerable interest in dissecting the various roles of
maternally and paternally derived imprinted
genes and other factors, including mitochondrial inheritance, in epigenesis (John and
Surani, 1996; Narasimha et al, 1997;
Latham, 1999).
Regarding non-germ-cell mitochondrial
transfer, there is a wide range of experimental
applications that are beyond the scope of
this review. There is, however, considerable
interest in the use of such systems as models
for approaches to curing or alleviating mitochondrial diseases and for studying mitochondrial importation mechanisms and drug
delivery systems (Chrzanowska-Lightowlers
et al, 1995; Murphy, 1997; Taylor et al,
1997, 1998; Clark et al, 1998). One relevant
and essential point is that the rapid evolution
of parts of the mitochondrial genome, in
concert with its partner nuclear genes, has
resulted in species-specificity for many mitochondrial functions. Thus, mtDNA from chimpanzees, bonobos, and gorillas could restore
oxidative phosphorylation to human mitochondrial-free cell lines, but mtDNA from
orangutan and Old and New World monkeys
and from lemurs could not (Kenyon and
Moraes, 1997). In these studies, oxygen consumption, a sensitive index of respiratory
function, indicated that mtDNA from the close
hominids restored mitochondrial respiration
and protein synthesis to normal levels. The
need for congruence between nuclear and
mitochondrial genes was highlighted by variations in embryonic metabolism and in
bioenergetic performance as adults of congeneric mouse constructs (Nagao et al., 1998a,b).
These considerations will have profound
implications for the development of cloning
technology as indicated below in the discussion on applications of cytoplasmic transfer
in human assisted reproductive technology.
Implications for human assisted
reproductive technology
There have been concerns expressed that intracytoplasmic sperm injection (ICSI) with
abnormal spermatozoa might lead to long-term
harm in the offspring through transmission of
abnormal mitochondria, although the evidence
for this so far is weak (Johns, 1995;
Houshmand et al, 1997; Cummins, 1998a).
There is evidence that spermatozoa even from
normal men contain significant levels of
mtDNA deletions (Cummins et al, 1998a) and
these may be elevated in men with poor semen
quality and infertility (Kao et al, 1995, 1998).
On a cautionary note, some authorities consider the evidence linking mtDNA deletions
and mutations with ageing (as opposed to
disease) to be poor and to be based on nonquantitative measure (Lightowlers et al,
1999). We have found, using competitive PCR,
that conventional semi-quantitative PCR
approaches (Zhang et al, 1996) can grossly
overestimate (up to 1000-fold) levels of deletions in rat mtDNA (Ahmed et al, 1999) and
in human mtDNA (D.Mehmet, unpublished
observations). On balance, however, there
seems to be little risk in using abnormal or
even immature sperm cells for ICSI, at least
with regard to transmission of abnormal
mtDNA. As discussed earlier, the paternal
mitochondrial line seems irreversibly committed to suicide in the embryo and any leakage
of abnormal mitochondria would be rare and
probably rapidly eliminated by natural selection during embryogenesis. There is probably
more risk of transmitting underlying genetic
disease and fragmented nuclear DNA, as men
with severe infertility may possess significantly increased levels of chromosomal and
meiotic abnormalities (Retief, 1986; Cummins
andJequier, 1995; Cummins, 1998b; Cummins
and Jequier, 1998; Vendrell et al, 1999).
Of greater concern are attempts by clinics
to 'rescue' poor quality oocytes and embryos
97
J.M.Cummins
by cytoplasmic transfer (Cohen et al., 1997,
1998) or nuclear transfer (Zhang et al., 1999).
These must be seen as risky and highly experimental procedures. Despite a long history of
experimental embryology, we still understand
very little about the balance between nuclear
and mitochondrial genes and the relative roles
of paternally and maternally derived nuclear
genes in development (John and Surani, 1996;
Narasimha et al, 1997; Latham, 1999). Moreover, as outlined above, it is not currently
possible to control the final fate of the donated
mitochondria in relation to nuclear-mitochondrial interactions or the embryonic axes, so
the outcome of donation would be highly
unpredictable. Similar problems beset the creation of heteroplasmic mice by karyoplast and
cytoplast fusion, the only good animal models
currently available (Meirelles and Smith,
1997, 1998).
Acknowledgements
I thank Adam Eyre-Walker, Erica Hagelberg, Robert
Jansen, David Keefe and Keith Latham for allowing me
to quote work in progress. In particular I wish to give
especial thanks to Peter Sutovsky for generously sharing
his thoughts and data on ubiquitin. My research on
mitochondrial DNA was supported by the Australian
National Health and Medical Research Council, in collaboration with Anne Jequier, Jack Goldblatt, Roger
Martin, Denise Mehmet and Jim Whelan. I also thank
Professor Ryuzo Yanagimachi for his hospitality and
for providing research facilities during visits to the
University of Hawaii.
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