Reviews of Reproduction (1998) 3, 172–182

Mitochondrial DNA in mammalian reproduction

Jim Cummins
Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, Western Australia 6150,
Australia

Mitochondrial DNA (mtDNA) forms a semi-autonomous asexually reproducing genome in

eukaryotic organisms. It plays an essential role in the life cycle through the control of energy
production, by the inherently dangerous process of oxidative phosphorylation. The asym-
metric nature of its inheritance – almost exclusively through the female – imposes different
evolutionary constraints on males and females, and may lie at the heart of anisogamy. This
review examines the implications of recent findings on the biology of mtDNA for reproduction
and inheritance in mammals.

Although the existence of mitochondria has been known since
the last century, mitochondrial DNA (mtDNA) has been studied
most extensively in the past two decades. Mitochondria have
a profound role to play in mammalian tissue bioenergetics, in
growth, in ageing and in apoptosis, and yet they descend from
an asexually reproducing independent life form. It has been
hypothesized that tensions between the evolutionary ‘interests’
of the eukaryotic host and its subservient organelles have led to
asymmetrical inheritance, so that mitochondria derive pre-
dominantly from the female in most organisms (Hurst, 1992;
Hurst et al., 1996). This has consequences that are only just be-
coming apparent. There is considerable interest in the potential
role of mitochondria and cytoplasmic inheritance on growth
and performance factors such as muscle development and milk
production in domestic animals. As might be expected, the
maternally inherited mitochondrial genome has significant
non-Mendelian effects on steroidogenesis and on respiratory-
dependent functions such as growth, oxygen consumption and
lean:fat ratios, but not on anaerobic metabolism. Smith and
Alcivar (1993) have reviewed this topic recently and compre-
hensively; therefore, the focus of this article will be the role of
mtDNA in the life cycle.
Mitochondria are semi-autonomous organelles found in all
eukaryotic cells (except mature red blood cells and some pro-
tozoans). It is generally accepted that mitochondria originated
in ancestral eukaryotic cells through endosymbiosis of free-
living bacteria capable of metabolizing oxygen – a suggestion
first made over a century ago and noted by Ozawa in his review
(Ozawa, 1997a). Our ancestral eukaryotes thus exploited the
capacity of mitochondria to metabolise oxygen. This allowed
them to flourish despite the increasing concentrations of this
highly reactive and potentially poisonous element in the en-
vironment. While most of the mitochondrial genes have moved
to the nucleus, mitochondria retain their bacterial facility for
multiplying by simple fission – and even fusing – indepen-
dently of the host cell cycle. Excess mitochondria are removed
by autophagic lysosomal activity. These population control
measures act in response to the energetic demands of different
tissues. The mitochondrial genome also has idiosyncrasies in
RNA processing and in its genetic code that differ from those of
nuclear DNA (for example UGA is normally a stop codon, but
© 1998 Journals of Reproduction and Fertility
1359-6004/98 $12.50
encodes for tryptophan in mammalian mitochondria). The in-
heritance of mitochondria through the female lineage remains
one of the central enigmas of reproductive biology.
Mitochondria have certain tissue-specific configurations that
presumably reflect local energetic requirements (Fawcett, 1981).
New techniques for visualizing mitochondria in whole cells,
such as the incorporation of green fluorescent protein coupled
with confocal microscopy, reveal that mitochondrial form can
not only reflect pathological states but may also be extremely
diverse even within the same cell (Kanazawa et al., 1997). The
basic design is a double membrane surrounding an inner mito-
chondrial matrix that contains one or more circular mtDNA
molecules. The outer mitochondrial membrane is thought to
represent the original invaginated host plasma membrane,
while the inner mitochondrial membrane represents the bac-
terial wall and contains the site of oxidative phosphorylation
(OXPHOS) on its inner surface. The matrix also contains ribo-
somes for local protein synthesis.
The two membranes differ profoundly in composition. The
lipid:protein ratio of the outer membrane is about 50:50 and
it is permeable to molecules with molecular weights of up to
10 000 (Lodish et al., 1995). The inner membrane is relatively
impermeable and is about 80% protein and is thrown up into
infoldings – crystae – the sites of OXPHOS enzymes. Here the
oxidation of metabolites generates ATP through a series of
integral membrane multi-subunit protein complexes which
couple electron transport to ATP synthesis. These complexes
are unique in that they consist of proteins encoded by two
separate yet cooperating genomes, that of the nucleus and that
of the mitochondrion (Poyton and McEwen, 1996; Shadel and
Clayton, 1997) There are separate translocase systems in the
inner and outer membranes that coordinate the recognition,
import and assortment of essential proteins from the cytosol
(Neupert, 1997).
Mitochondrial DNA
Most cells in the body contain between 103 and 104 copies of
mtDNA. There are much higher copy numbers (about 105) in
mature oocytes. This may be in preparation for the energetic
demands of embryogenesis (Pikó and Matsumoto, 1976) but
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 Mitochondrial DNA in the life cycle
Table 1. Fate of light and heavy chain transcripts from mitochondrial
DNA
Light chain transcripts Heavy chain transcripts
8 tRNAs 14 tRNAs
1 mRNA 12 mRNAS
RNA primers for heavy chain replication 2 tRNAs
Modified from Shadel and Clayton, 1997
an alternative explanation is that replication does not occur
during early embryogenesis and that high copy numbers are
needed to give a sufficient reservoir (see below). The DNA
exists mainly as a circular molecule of approximately 16.6 kb,
encoding 13 proteins that are transcribed and translated in
the mitochondrion (Table 1). These are essential subunits of the
electron transport complexes on the inner mitochondrial mem-
brane. The mitochondrial genome also encodes the RNA mol-
ecules that are necessary for the translation of these proteins
(Table 1) (Lodish et al., 1995; Shadel and Clayton, 1997).
mtDNA structure
The separate strands of the mtDNA molecule differ in buoy-
ant density; the heavier ‘H-strand’ has a higher G + T content
than the light ‘L-strand’. Transcription occurs simultaneously
and in opposite directions and many genes overlap. By con-
vention mtDNA is depicted as a circle (Fig. 1), but alternatives,
such as dimer loops and catenated (chain-linked) circles, are
known (Clayton, 1982). In some single-celled organisms (many
pathogenic to mammals) aberrant linear mtDNA molecules
with telomere-like endings are also found (Nosek et al., 1998).
There is a specialised, somewhat unstable and hypervariable
region called the D-(displacement) loop, where there is a triplex
DNA structure at the site of origin of the H strand (Fig. 1). This
structure is formed by a short nascent H-strand that remains
closely associated with the parental molecule. This region is crit-
ical for the initiation of transcription and translation (see below).
The mammalian mitochondrial genome is extremely com-
pressed with no introns. Some genes even overlap. This con-
trasts strikingly with, for example, yeast (five times larger at
78 000 bp) or plants, in which there are multiple recombining
molecules with enormous size variation even within a family.
For example, sizes vary from 330 000 bp in watermelons to
2.5 + 106 bp in muskmelon (Lodish et al., 1995). Most of the 100
or more genes controlling the synthesis of mammalian mito-
chondrial proteins have moved to the nucleus over the course
of evolution.
Import–export mechanisms
Proteins are assembled on cytoplasmic ribosomes and trans-
ported into the mitochondrion through protein-lined channels,
with the aid of cytoplasmic chaperone proteins (Box 1). These
import mechanisms are prime targets for strategies aimed at
mitochondrial genome therapy or modification (Murphy, 1997;
Taylor et al., 1997). Import involves hydrolysis of ATP in both the
cytoplasm and the mitochondrion plus an electrochemical poten-
tial across the inner mitochondrial membrane (Lodish et al., 1995;
173
Displaced heavy strand (D-loop)
Heavy strand Nascent heavy
origin strand
Heavy strand
promoter
Transcription
factor binding Light strand
sites promoter
Light strand origin
(stem-loop)
Fig. 1. This diagram summarizes the major features of mtDNA re-
ferred to in the text. (See Shadel and Clayton, 1997.)
Box 1 Major mitochondrial import proteins
Mitochondrial DNA and RNA polymerases
Transcription, translation and transcription termination factors
RNA processing enzymes
Mitochondrial ribosomal proteins
Aminoacyl-tRNA synthetases
Neupert, 1997). Besides structural components of the mitochon-
dria, these imports involve factors that regulate and specifically
recognize mtDNA and regulate gene expression (Box 1).
Nuclear–mitochondrial interactions
Any alterations that arise in the components of the mtDNA
or RNA that recognize or bind to nuclear-encoded regulatory
elements must be balanced by compensatory mutations in the
nuclear genes, as the mitochondrial genome mutates much
more rapidly than the nuclear genome (see below). This mu-
tuality is thought to drive species specificity in nuclear–
mitochondrial interaction (Kenyon and Moraes, 1997; Wallace,
1997), but surprisingly little is known about the coordination of
expression between the nuclear and mitochondrial genomes.
However, it is clearly a sensitive system. Nagao et al. (1998)
found decreased physical performance and growth rates in
mice in which the nuclear and mitochondrial genes were mis-
matched. The mitochondrial genome exists as multiple
copies per cell and yet for unknown reasons proteins encoded
by single copy nuclear genes are present in the same amounts
as mtDNA-encoded proteins (Poyton and McEwen, 1996). This
is clearly an issue that will bear on attempts to clone animals,
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174 J. Cummins

Table 2. Summary of some major mtDNA functional locations in the mtDNA. While the ‘common’ deletion is the most prevalent,
human mitochondrial genome this is also practically convenient as the major difference in
size between the deleted and the intact mtDNA gives an un-
Nucleotide position* Description ambiguous signal after PCR amplification. With ageing the
genome undergoes extensive fragmentation. One study still
110–441 H-strand origin awaiting independent verification suggests that in very old
233–260 mtTF1 (=Tfa) binding site human individuals the amount of wild-type intact mtDNA
276–303 mtTF1 (=Tfa) binding site may be as low as only 11% of the total (Hayakawa et al.,
299–315 Conserved sequence box (CSB) II 1996). The mtDNA in these cases can fragment into mini-circles
317–321 Replication primer lacking replication origins; if and how these replicate is
346–363 CSB III unknown.
371–379 mt4 H-strand control element Life expectancy within a species is closely correlated with
384–391 mt3 H-strand control element overall nuclear DNA repair efficiency and inversely correlated
392–445 L-strand promoter with mtDNA damage; for example mtDNA mutation and
418–445 mtTF1 (=Tfa) binding site general body tumour formation rates are approximately 40 fold
523–550 mtTF1 (=Tfa) binding site higher in mice than in humans (Cortopassi and Wang, 1996).
545–567 Major H-strand promoter Although mitochondria obviously play only a part in the ageing
645–645 Minor H-strand promoter story (Holliday, 1995), it seems clear that they hold many of the
5721–5798 L-strand origin keys to life cycle dynamics, through their mode of inheritance
15925–15499 Membrane attachment site and through tissue-specific control of energy production.
16194–16208 Control element
16499–16506 L-strand control element Replication and transcription
16157–16172 Termination sequence
Although the topology and major events of mitochondrial
(From Mitomap URL http://www.gen.emory.edu/cgi-bin/MITOMAP/bin/ replication and transcription are well established (Shadel and
tbl1gen.pl). Clayton, 1997), their differential control is still rather poorly
*Nucleotide positions are based on the published sequence of human mtDNA by understood. Transcription is from each strand, starting from
Anderson et al. (1981). light and heavy chain promoter regions (Table 2). This is bi-
directional, resulting in the production of large polycistronic
where there may be lack of complementarity between nuclear transcripts that are later processed to form mature RNAs
and mitochondrial genomes that may affect body weight (Table 1).
and performance (Gartner et al., 1998). Nuclear–mitochondrial Although there is interaction between the mitochondrial and
interactions will be discussed later with replication and nuclear genomes, mtDNA replication and mitochondrial div-
transcription. ision are clearly uncoupled from the nuclear division cycles.
The two strands of mtDNA have two separate origins for
replication. That for the H-strand (OH) is within the D-loop,
Function in tissue bioenergetics and ageing
whereas that for the L-strand (OL) is about one third of the way
Mitochondria largely control the production of energy by around the genome (Fig. 1). Replication is dependent on the
oxidative phosphorylation (OXPHOS). Defects caused by in- binding of the nuclear encoded transcription factor A to its
herited or cumulative mutations and deletions are now seen as binding site or sites (Table 2). This triggers transcription from
central to many disease states as well as apoptosis and the OH by the core mtRNA polymerase to form an RNA–DNA
ageing process (Shoffner and Wallace, 1990; Ozawa, 1997a; hybrid, involving some subset of the conserved sequence
Wallace, 1997). The mitochondrial genome is vulnerable to blocks (CSBs). The RNA–DNA hybrid acts as a template for as-
oxidative attack as it lies at the site of the electron transfer chain sembly of a new H-strand by DNA polymerase gamma.
(the site of intensive generation of reactive oxygen species) and Replication proceeds in a clockwise manner and when it
lacks protective histones. Consequently it mutates ten or more reaches position 5721–5798, OL is exposed and replication of
times faster than nuclear DNA. Mutations can take the form of the L strand commences in the opposite direction. The lag in
single or multiple base pair replacements or modifications. replication results in the formation of two daughter molecules,
Replication errors can also generate deletions by internal re- conventionally termed a and b: b consists of the original H
combination, generally around tandem repeat ‘hot spots’. strand plus a partially replicated L strand. This proceeds to
Deletions and mutations accumulate preferentially in post- complete replication of the L strand before circle closure.
mitotic tissues such as the heart and the central nervous Completion of replication involves supercoiling and the syn-
system. This is because, in rapidly dividing tissues, cell lines thesis of a new H strand. The whole process is very slow (E. coli
that accumulate lethal threshold concentrations of mtDNA replication is about 200 times faster), taking approximately 2 h
dysfunction can be eliminated: a form of cytological natural to complete. Replication is also prone to error; deletions of
selection. Post-mitotic tissues have no such way of eliminating large segments occur especially at regions of tandem repeat
‘bad’ mitochondria, which accumulate and eventually lead to ‘hot spots’ (Ozawa, 1997a).
irreparable damage. Laboratories attempting to quantify Replication and transcription appear to be differentially
mtDNA damage generally concentrate on major deletions regulated, and there are a number of regulator and promoter
such as the ‘common’ 4977 base pair deletion seen in human sites on the genome (Table 2).

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 Mitochondrial DNA in the life cycle 175

Tissue and species specificity not generally transmitted, there is no long-term selective ad-
vantage for mtDNA to be healthy in males – from the ‘selfish
The exact mechanisms by which different tissues control
gene’ point of view, the fitness of the male mitochondrial
their mitochondria are still being debated (Shadel and Clayton,
genome is irrelevant. One could even argue a scenario in which
1997). Replication and transcription do not appear to be regu-
a maternally inherited defective mtDNA in a man may inhibit
lated by availability of DNA polymerase, as expression of the
his fitness and thus allow his sisters to flourish. At least one of
catalytic subunit of DNA polymerase gamma varies little
the mitochondrial DNA diseases (Leigh’s Hereditary Optic
among different tissues and is not increased in muscle mito-
Neuropathy, LHON) is more prevalent and occurs earlier in
chondrial biogenesis after motor nerve stimulation. One
males than in females, which reinforces this hypothesis (Johns,
possibility for control is through the nuclear-encoded RNA
1995). Moreover the free lifespan of spermatozoa, exposed to
polymerase that recognizes promoters in a species-specific
enormous ranges in oxygen tension (Max, 1992), coupled with
manner (Schultz et al., 1998) This could be the mechanism by
poor antioxidant defence systems leaves the mtDNA of the
which higher primate mtDNA can substitute for human
spermatozoa highly vulnerable to damage. There is evidence
mtDNA in a depleted cell (see below), as there are high degrees
that some forms of male infertility may be associated with
of conservation in the D-loop sequences for the promoter re-
defects in mitochondrial function and testicular OXPHOS
gions and for mtTF1 (Kenyon and Moraes, 1997). It is signifi-
(Cummins et al.,1994a).
cant that mtDNA replication within the cell begins close to
the nucleus as this clearly demonstrates the importance of
nuclear-derived signals for activating this process (Davis and Mitochondria in spermatogenesis
Clayton, 1996). Larsson et al. (1998) used targeted gene
It is necessary to trace the fate of the mitochondria in sper-
knockout in mice for the gene for mitochondrial transcription
matogenesis to form a working hypotheses on why the mito-
factor A (mtTFA now renamed Tfam). Heterozygous embryos
chondria from spermatozoa are not generally transmitted after
show reduced mtDNA copy number and respiratory chain
sperm entry (Clermont et al., 1993; Hecht, 1995).
deficiency in cardiac muscle, but homozygous knockout em-
The mitochondria that populate the primordial germ cells
bryos have severe depletion of mtDNA and no OXPHOS and
and the support elements of the testis (Sertoli, Leydig, peri-
die before day 10.5.
tubular myoid and other connective tissue cells) are typical of
The development of ‘clones’ such as the sheep Dolly
somatic cells. With the onset of meiosis, the germ cell mito-
(Wilmut et al., 1997) produced by nuclear transfer highlight the
chondria start to differentiate and the mitochondrial matrix be-
need for understanding the relationship between the nucleus
comes diffuse and vacuolated. Differentiating spermatids also
and the cytoplasm. There is clearly a degree of species spec-
lack heat shock protein 60 (hsp60), a mitochondrial chaperonin,
ificity in mitochondrial function. Kenyon and Moraes (1997)
which suggests that they may also be deficient in protein im-
used a human cell line depleted in mtDNA (Rho 0) and found
port mechanisms (Meinhardt et al., 1995). These ‘germ cell’
that mtDNA from gorillas, chimpanzees and pygmy chim-
mitochondria finally make up the majority (> 80%) of those
panzees could substitute for human mtDNA in supporting res-
found in the testes of mature animals. The mitochondria cluster
piration and mitochondrial protein synthesis, while mtDNA
near the nucleus during meiosis and spermiogenesis and then
from orangutans, and species representative of Old World
differentiate progressively into the crescentic mature sperm
monkeys, New World monkeys and lemurs could not. Within
type. Some proliferation may occur early in spermiogenesis,
species it also appears that there may be factors that select for
as indicated by mtDNA replication, but copy numbers later
and against individual mtDNA types. Jenuth et al. (1997) found
decline so that each mature sperm mitochondrion contains, on
tissue-specific and age-related variations in mtDNA genotype
average, only one copy of mtDNA. However there is no evi-
survival in heteroplasmic mice, suggesting that individual tissue
dence of any gross alteration or selective methylation of the
energetic demands could bias the survival or segregation of
sperm mtDNA that could explain its later loss in the embryo
different mitochondrial genomes. Meirelles and Smith (1997)
(Hecht and Liem, 1984; Hecht et al., 1984; Hecht, 1997). The
found significant variation in the segregation of different mito-
midpiece is delineated by caudal migration of the annulus and
chondrial genomes in mice produced by karyoplast fusion.
then mitochondria migrate along the axoneme in a controlled
However, some of this may have been due to the placement of
stepwise fashion to form a tight array (Otani et al., 1988).
exogenous mitochondria in relation to the nucleus and to em-
Differentiation of the sperm mitochondria into a crescentic
bryonic axes (see below). Nagao et al. (1998) have shown sig-
shape appears to be pre-programmed, as it also occurs in
nificant depression of OXPHOS-dependent exercise capacity in
the superfluous mitochondria of the excess cytoplasm.
interspecific and inter-subspecific congenic mice produced by
Moreover, it appears to depend on specific interaction with
repeated backcrossing, pointing to poor coordination between
the Sertoli cell (Seitz et al.,1995). Formation of the midpiece is
the mismatched nuclear and mitochondrial genomes. The
accompanied by the secretion of a selenoprotein-based mito-
question of species-specific recognition of mitochondria by the
chondrial sheath capsule and a sub-mitochondrial reticulum
host cell returns when inheritance is considered.
that anchors the sheath to the axoneme proper (Olson and
Winfrey, 1992). The capsule consists of at least four proteins
ranging from 17 to 31 kDa (Cataldo et al., 1996). Presumably
Why is there maternal inheritance of mtDNA ?
this structure conveys mechanical strength, as the sperm
Males, like females, receive their mtDNA from their mother. mitochondria are relatively resistant to swelling under
This unequal mode of inheritance may have consequences for osmotic stress compared with other cytoplasmic components
health and fertility. As the mitochondria of spermatozoa are (Willoughby et al., 1996). Spermatozoa contain the highest

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176 J. Cummins
relative concentration of selenium of any cell line in the body,
and animals fed a selenium-deficient diet show midpiece
defects (Cataldo et al., 1996). The protein is probably the anti-
oxidant, phospholipid hydroperoxide glutathione peroxidase
(Roveri et al., 1994), which is intriguing given the known
susceptibility of spermatozoa to oxidative damage and lipid
peroxidation (Aitken, 1995).
Other sperm-specific unique mitochondrial proteins also
appear during spermiogenesis. These include a sperm-specific
lactic dehydrogenase isoenzyme that appears early (Machado
de Domenech et al., 1972) and a unique isoform of cytochrome c
(Goldberg et al., 1977). The spermatid nuclei lose the mito-
chondrial targeting sequence of a testis-specific isoform of nu-
clear mitochondrial transcription factor A (Larsson et al., 1994,
1997), suggesting that there is no need for mitochondria to
replicate once on the midpiece. However, the mitochondrial
transcriptional potential of the spermatozoon is apparently un-
changed during this process (Alcivar et al., 1989). Larsson et al.
(1996) suggest that the nuclear isoform of mtTFA, which is also
present in spermatocytes, plays a secondary structural role in
compacting nuclear DNA during spermiogenesis. This would
be consistent with its known capacity to induce conformational
changes in DNA (Fisher et al., 1992).
Role of mitochondria in sperm function
Although the mitochondrial sheath of spermatozoa clearly
has a functional role, rather surprisingly there is little consen-
sus about the relative importance of mitochondrial respiration
(as opposed to glycolysis) for motility, or even for fertilization
itself (Bedford and Hoskins, 1990). Spermatozoa in which the
mitochondria have been poisoned with cyanide can induce
normal embryo development when microinjected into oocytes
(Ahmadi and Ng, 1997). Spermatozoa are metabolically flexible
and, in some species, can switch between aerobic and anaerobic
metabolism. This perhaps reflects the great range of oxygen
tensions they experience, from near anoxia in the testis and
epididymis to ambient tensions in the vagina and in vitro
(Ford and Rees, 1990; Max, 1992; Cummins et al., 1994b). New
techniques, such as flow cytometry using vital dyes and fluoro-
chromes, that can indicate the degree of mitochondrial mem-
brane potential promise to elucidate the relationship between
mitochondrial function, overall sperm viability and fertilizing
potential (Garner et al., 1997). Like somatic mtDNA, that of
spermatozoa is highly vulnerable to mutation, and a significant
number of mtDNA deletions are found in the semen of at least
50% of normospermic men (Cummins et al., 1998). Given the
lengthy process of spermiogenesis and epididymal maturation
during which the sperm mitochondria have to survive without
any interaction with the nuclear genome, and given the likeli-
hood that they will be exposed to mutagenic agents, this is per-
haps not surprising. Indeed, the need to exclude defective
sperm mtDNA from contributing to the embryo is possibly one
of the major selection pressures against survival of paternal
mtDNA. Indeed Short (1998) has suggested that this asym-
metric inheritance of mtDNA, through the oocyte but not the
spermatozoon, may be a fundamental driving force behind
amphimixis and anisogamy. This is because of the need to con-
serve a healthy stock of mtDNA for embryo development,
through a long period of quiescence in meiosis.
What happens at fertilization?
A flood of misinformation in undergraduate textbooks and
popular texts is promoting the ‘African Eve’ model of human
evolution and minimizes and even denies the possibility of
paternal mtDNA transmission (Ankel-Simons and Cummins,
1996). At least one well-respected author (Richard Dawkins)
has claimed (but later retracted) that spermatozoa are too small
to contain mitochondria, a statement that comes as a surprise to
most biologists (Dawkins, 1986, 1995). In fact, total sperm entry
into the egg including the midpiece mitochondria is the rule
rather than the exception for most mammals. However, it is
now well established that the mitochondria from spermatozoa
are targeted for destruction by endogenous proteolytic activity
during early embryogenesis (see below). Uniparental (gener-
ally but not universally maternal) inheritance of cytoplasmic
organelles like mitochondria and, in plants, plastids, is ac-
complished by a wide variety of strategies and thus is clearly of
profound importance to long-term fitness. It is usually argued
that uniparental inheritance in sexually reproducing organisms
minimizes the risk of lethal genomic conflict between asexually
reproducing cytoplasmic genes (Hurst, 1992, 1995; Hurst et al.,
1996) but how this is accomplished remains one of the major
puzzles for evolutionary reproductive biology. Indeed Birky
(1995) states that “... the variety of molecular and cellular mech-
anisms found in different organisms is matched only by the
variety of hypotheses devised to explain the evolution of
the phenomenon”. Perhaps the most bizarre example is in the
giant spermatozoa of some species of Drosophila, in which
the male mitochondria are sequestered to the midgut during
organogenesis and defaecated by the larvae soon after hatching
(Pitnick and Karr, 1998).
Paternal transmission – an anomaly?
Most of the evidence indicating the possibility of paternal
transmission of mtDNA derives from interspecific crosses,
which by definition are uncommon in nature (Kaneda et al.,
1995). Backcrossing studies in Mus spretus + Mus musculus
hybrids showed a ‘leakage’ of paternal mtDNA of about 1 in
1000 to 1 in 10 000 molecules (Gyllensten et al., 1991). This is not
far from the theoretical ratio of oocyte to sperm mtDNA seen at
fertilization (Ankel-Simons and Cummins, 1996) but this obser-
vation of leakage may be an artefact (see below). Kaneda et al.
(1995) investigated this question in an ingenious manner, using
a nested polymerase chain reaction (PCR) assay that could de-
tect sperm mtDNA in a single mouse embryo. In embryos pro-
duced by in vitro fertilization of Mus musculus eggs with the
same strain of spermatozoa, paternal mtDNA could be detected
in zygotes but only at the early pronuclear stage, and its dis-
appearance coincided with loss of membrane potential in
sperm-derived mitochondria, as measured by Rhodamine 123
fluorescence. By contrast, when Mus spretus spermatozoa were
used to fertilize M. musculus eggs, paternal mtDNA was de-
tected at the pronucleus stage, at the two-cell stage and in
neonates. This was confirmed by sequencing the PCR product.
When spermatozoa from the congenic strain b6.mt(spr), which
carries M. spretus mtDNA on a background of M. musculus
nuclear genes, was used, the M. Spretus mtDNA was elimi-
nated early in development, as in intraspecific crosses (Kaneda
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 Mitochondrial DNA in the life cycle
et al.,1995). Kaneda et al. (1995) proposed that the zygote cyto-
plasm has a species-specific mechanism that recognizes and
eliminates sperm mitochondria, on the basis of nuclear DNA
encoded proteins in the sperm midpiece, and not on the
mtDNA itself, nor on the proteins it encodes. In further work,
the same group found that the ‘leaked’ paternal mtDNA fol-
lowing interspecific crosses is limited to the first generation,
does not get transmitted to all tissues and does not survive
into subsequent back-crosses (Shitara et al., 1998). This calls into
question the original findings of Gyllensten et al. (1991) and
suggests that the congenic mouse strain they used in detecting
paternal leakage may have retained a stable heteroplasmy. This
may have arisen from mitochondrial fusion as is thought to
have occurred in at least one line of mice produced by cytoplast
transfer (Meirelles and Smith, 1997).
Although this picture appears convincing, there are some
problems with these data and the results have not been repli-
cated by other laboratories. First, nested PCR detection of very
small amounts of DNA is fraught with problems of potential
contamination, and most laboratories insist on dual facilities
with double-blind controls when carrying out procedures such
as embryo sexing. It is not clear whether such precautions
were taken here. Second, the authors observed that midpiece
Rhodamine fluorescence disappeared at the late pronuclear
stage in intraspecific embryos, indicating loss of mitochondrial
membrane potential. This is very different from our own obser-
vations using the stable fluorochrome MitoTrackerr (Cummins
et al., 1997) in which viable midpiece mitochondria were found
up to day 3 in four-cell embryos and up to day 5 in non-
activated oocytes and arrested two- and four-cell embryos.
By contrast, other sperm tail components, such as the coarse
tail fibres, can be identified up to the blastocyst stage and poss-
ibly even later. Clearly there is species-specific recognition and
destruction of the mitochondrial sheath, but it occurs later in
embryogenesis than was proposed by Kaneda et al. (1995).
Moreover the mitochondria in the interspecific-cross embryos
of Kaneda and co-workers (in which the paternal mtDNA per-
sisted) should have been visible by light microscopy, but the
authors made no comment on this. Findings in other rodent
species and cattle (summarized in Cummins et al., 1997) all in-
dicate independently that sperm mitochondria persist into the
second cell cycle before lysis. Sutovsky et al. (1996) proposed
that the mitochondrial sheath in cattle may be tagged with
ubiquitin for proteolytic destruction and they now have pre-
liminary evidence confirming this interesting idea (Sutovsky,
personal communication).
Are sperm mitochondria viable?
Single somatic cell mitochondria can colonise mtDNA-
depleted cell lines under experimental conditions in vitro, albeit
at low efficiency (King and Attardi, 1989). Manfredi et al. (1997)
found even lower long-term colonization when spermatozoa
were fused with mitochondrially depleted somatic cell lines
in vitro. Only 10–20% of cells had mitochondria demonstrating
normal transmembrane potential when examined 4 h after
fusion. One possibility is that the highly differentiated sperm
mitochondria lack functional protein import mechanisms and
therefore cannot respond to nuclear-encoded signals when they
receive them (Neupert, 1997). It is not yet clear whether eggs and
177
Table 3. Possible mechanisms for uniparental inheritance of mammalian
mitochondria
Mechanism Evidence in mammals?
Prezygotic
Anisogamy – disparity in numbers between Yes
eggs and spermatozoa
Exclusion of mitochondria from spermatozoa No
Degradation of mitochondria in spermatozoa No
Degradation of mtDNA in spermatozoa Probable
Fertilization
Exclusion of sperm mitochondria from zygote Very rare
Zygotic/deterministic
Selective degradation of sperm mitochondria Yes
Selective degradation of sperm mtDNA No
Targeted partitioning of sperm mitochondria Remotely possible
into non-embryonic cells
Zygotic/stochastic
Random partitioning of sperm mitochondria Remotely possible
into non-embryonic cells
embryos have a generalized defence as opposed to an intra-
specific defence against exogenous mitochondria. Hecht (1984)
was unable to detect microinjected conspecific testicular and
liver mitochondria in offspring but, more recently, transmission
of mutant human mtDNA (from an individual who died of
mitochondrial disease) has been reported in mouse embryos
(Rinaudo et al., 1996). Pinkert at al. (1997) have also reported that
Mus spretus liver mitochondria microinjected into Mus domesticus
zygotes persist to the blastocyst stage. Such models will be very
valuable tools for the study of mtDNA disease transmission
and in the search for means to target and perhaps even cure
such disorders (Taylor et al., 1997).
How are sperm mitochondria recognized by the zygote?
At present the mechanism that recognizes and targets the
sperm midpiece for destruction is not known although there
is now evidence that it is mediated by ubiquitin (Sutovsky,
personal communication). This is one of the central mysteries
of reproductive biology. The key may lie in the asynchrony
between mtDNA transcription and replication in the embryo.
The simplest explanation (apart from dilution beyond the poss-
ibility of subsequent detection) is that the sperm mitochondria
cannot maintain membrane potential up to day 6.5 when repli-
cation of the mtDNA of the embryo begins (Pikó and Taylor,
1987; Ebert et al., 1988). Leakage of key components such as
cytochrome c, a known trigger for apoptosis (Ozawa, 1997b)
from the mitochondria, may signal multivesicular bodies to
surround the mitochondria and digest them (Hiraoka and
Hirao, 1988) Against this must be set the observations on
persistence of paternal mtDNA in interspecific mouse crosses
(Kaneda et al., 1995; Shitara et al., 1998).
Several other possibilities are summarized on the basis of
Birky (1995) (Table 3). The actual mechanism remains to be
elucidated fully.
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178
Embryogenesis, oogenesis and the
‘restriction–amplification’ model of mtDNA transmission
With very few exceptions such as mussels, most organisms
appear intolerant of heteroplasmy for mtDNA, even though
stable heteroplasmic lineages can be created artificially (Jenuth
et al., 1996, 1997). When it does occur it generally arises by
de novo mutation during oogenesis and in humans is frequently
associated with progressive mitochondrial bioenergetic dis-
eases in offspring. Many homoplasmic mutations are probably
lethal in early embryogenesis owing to oxidative phosphoryl-
ation insufficiency (Ozawa, 1997a; Wallace, 1997). Neutral
mutations that arise can become fixed in the germline very
rapidly – within one to three generations (Ashley et al., 1989).
One possibility is that this may arise through a restriction–
amplification process during the exponential growth phase of
oogenesis, in which mitochondria multiply by division from a
few precursors (Marchington et al., 1997; Nagley et al., 1993).
Blok et al. (1997) even suggested that the progenitor pool could
be a single mitochondrion in humans. During oogenesis, this
pool expands considerably resulting in about 105 mitochondria
in the mature oocyte (Pikó and Taylor, 1987).
Alternatively, Jenuth et al. (1996, 1997) suggest that the ‘bottle-
neck’ may be an artefact caused by random genetic drift during
early embryogenesis. By contrast, Steinborn et al. (1998a,b) found
that the ratio of different mtDNAs found in cattle, produced by
fusion of nucleated blastomeres with enucleated metaphase IIm
oocytes, was closely related to the stage of the donor embryo:
13–18% when the donor was a 24-cell morula and 0.4–0.6% when
the donor was a 92-cell morula. Thus there was no doubt that
donor embryo mtDNA can persist in such heteroplasmic clones.
This hypothesis is currently contentious. An anonymous
reviewer of the present paper stated “The clonal expansion of
a limited pool of mtDNA precursors could be likened to an
urban myth: there is no direct evidence for it, and perhaps no
need to propose it”. One trusts that the myth, if it is one, will
soon be resolved.
Mitochondria in oogenesis and axis formation in embryogenesis
The mammalian oocyte contains 2 × 105 copies of the mito-
chondrial genome in approximately 100 000 mitochondria.
In immature oocytes these appear to be evenly distributed;
however, Calarco (1995) found that a distinct polarity appeared
after meiosis re-started. A cortical group of mitochondria
marks the extrusion point of the first polar body. Other periph-
eral foci of mitochondria tend to be concentrated in the hemi-
sphere containing the metaphase II spindle. New observations
on the distribution of leptin and STAT3 (Antczak and Van
Blerkom, 1997) demonstrate that the axes of the oocyte and
resulting embryo are determined very early – perhaps by pos-
itioning in relation to blood flow and oxygen availability in the
follicle. Leptin is the 16 kDa cytokine product of the obese gene
(ob) and activates STAT3, one of the Signal Transducer and
Activation of Transcription family of proteins. The first cell div-
ision is meridional resulting in two cells with equal leptin/
STAT3 gradients. However, the second cell cycle has one
meridional and one equational division. This results in one
blastomere being leptin/STAT3 deficient and this cell goes
on to form the inner cell mass (ICM) of the blastomere (Fig. 2).
J. Cummins
The role of leptin in the embryo is obscure but presumably it is
involved with resource allocation between the ICM and the
trophectoderm. Clearly our ideas about the ‘totipotency’ of
the individual embryo cells up to the eight-cell stage need to
be revised in a major way (Edwards and Beard, 1997).
That there is significant polarity in the oocyte and embryo
may help to explain the skewed segregation of mitochondrial
genotypes in heteroplasmic mice created by cytoplast or
karyoplast fusion. When mitochondria were placed in the
periphery of oocytes (cytoplast transplantation) the resulting
mice showed significantly more variation than when mito-
chondria were placed near the (karyoplast derived) nucleus
(Meirelles and Smith, 1997, 1998). As it is not possible to place
cytoplasts accurately to the primordial oocyte axis before
electrofusion there can be less certainty that they will end up in
the ICM and hence in the somatic (and germ) cells of the
embryo. Another factor to consider is that mitochondrial repli-
cation starts in regions of the cytoplasm close to the nucleus
(Shadel and Clayton, 1997), so spatial positioning may affect
mitochondrial transmission.
In mice the amount of mtDNA – and presumably therefore
the number of mitochondria – does not change during develop-
ment up to the blastocyst and early egg cylinder stage (Ebert
et al., 1988). There are approximately 910 cells at day 6.5
(Hogan et al., 1986) when the primordial germ cells (PGCs)
first differentiate from the proximal epiblast, so if mitochondria
are randomly apportioned between cells during this time,
the founder PGC will receive approximately 100 founder
mitochondria. If mitochondria are not equally segregated to
this cell line, the number could be even smaller (Olivo et al.,
1983; Ashley et al., 1989). This is a similar order of magnitude
to the numbers in oogonia proposed by Jenuth et al. (1996)
(see above). It is possible that only follicles containing oocytes
with a high degree of homoplasmy are permitted to achieve
dominance. One way this could be tested would be to evaluate
OXPHOS parameters and mtDNA integrity in dominant com-
pared with atretic oocytes.
Mitochondrial DNA in the ageing ovary
Unlike spermatogenesis which is continuous from puberty,
oogenesis starts during fetal life (Baker, 1982; Byskov, 1982).
Oogonia proliferate by mitosis, reaching a peak in mid-
gestation. In most mammals, oogonia enter meiosis at this time
but arrest at dictyotene. Oocyte growth resumes with recruit-
ment of primordial follicles during the ovulatory cycle and
meiosis normally completes only at about the time of ovulation
and fertilization. Thus there is an extended time (50 years or
more in women) when the nuclear and cytoplasmic integrity of
the oocyte need to be maintained without cell division. Atresia
at all stages means that the vast majority of oocytes never make
it to ovulation: of the 7 million found at mid-gestation in
humans only a few hundred will ever reach ovulatory potential
(Baker, 1982). Lactational amenorrhoea coupled with birth
spacing of about four years in hunting–gathering societies
means that probably only 10–15 oocytes were ever released in a
woman’s life while humans were evolving. Even the menstrual
cycle may have developed as a non-adaptive consequence of
uterine evolution (Finn, 1998). Not surprisingly, the fertility
of individual oocytes declines with age along with reduced
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 Mitochondrial DNA in the life cycle 179

Fertilization

Two-cell
mtDNA transcription
starts

Syngamy
Polarity established

Four-cell
Axis commitment
Spermatogenesis Sperm mitochondria
Oogenesis disappear
Reduction in mtDNA
Restriction/amplification copy number
of mtDNA Mitochondrial
Massive atresia differentiation
Prolonged dictyate Loss of replication
meiosis capacity?

Germ cell line

Tissue differentiation Day 6–7 – egg cylinder

Germ cell commitment
mtDNA replication starts
Somatic cell line
Random segregation of mtDNA to tissues
Tissue-specific selection?
Age-specific selection?
Age-related mutations/deletions
Critical threshold effects
Declining OXPHOS efficiency
Ageing and death

Fig. 2. This schematic life-cycle summarizes some of the major ways in which the life cycle of the eukaryotic host interlocks with that of its
mitochondria. The concepts of gradients and axis formation in the embryo are based on those of Antczak and Van Blerkom (1997). OXPHOS,
oxidative phosphorylation.

capacity of the reproductive tract to support pregnancy
(Adams, 1984). There is considerable interest in the potential
role of mtDNA in this process, together with the effects of
altered OXPHOS and cellular antioxidant systems on the cyto-
skeleton, fertilization and subsequent embryo development
(Tarin, 1996). However, the data will be hard to evaluate until
accurate methods for quantifying mtDNA deletions and point
mutations have been developed. Even normal human oocytes
can be shown to contain mtDNA deletions and rearrangements
using extensive PCR cycling (Chen et al., 1995; Keefe et al.,

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180 J. Cummins
1995). Kitagawa et al. (1993) found large numbers of deletions
in women aged 45 and over, and Müller-Hocker et al. (1996)
showed age-related declines in OXPHOS enzyme components,
and alterations in mitochondrial morphometry that could cor-
relate with declining fertility. These alterations could relate to
the reduced ATP content and potential for development in the
poorer quality oocytes and embryos found in older women
(Van Blerkom et al., 1995; Van Blerkom, 1997). This is a rich
potential area for future research, as we may find that oocyte
potential is defined by intra-ovarian factors such as blood
supply and local oxygen tension.
There are already reports of clinics attempting to improve
IVF results by donor cytoplasmic transfer in women with
poor quality oocytes and recurrent implantation failure (Cohen
et al., 1997, 1998). This was coupled in at least one case with re-
moval of cytoplasmic fragments from embryos, but in this case
mid-pregnancy testing revealed that the mtDNA was of the
mother not the donor, so that the benefits of manipulation
are unclear.
Why don’t mitochondria have sex?
Sex, or more specifically homologous recombination (HR) be-
tween mtDNA molecules, is assumed not to occur. Even when
there is clear evidence of heteroplasmy within the cytoplasm
there is no convincing evidence that HR occurs between popu-
lations (Hayashi et al., 1994). However, under some circum-
stances mitochondrial DNA may be capable of recombination
and short-term repair (Thyagarajan et al., 1996; Salazar and
Vanhouten, 1997; Anson et al., 1998). Howell (1997) reviewed
the evidence and concluded that “_ the physiological signifi-
cance of this recombinase activity remains inaccessible, because
there is still neither any evidence for reciprocal recombination
of mtDNA molecules nor any apparent reason that such an
activity would be beneficial.” Howell (1997) suggests that the
HR described by Thyagarajan et al. (1996) may be due to a
mistake in the topoisomerase–resolvase complex that separates
and re-seals daughter mtDNA molecules at replication. This is
an imperfect process that frequently results in concatenated
oligomers and other configurations (Howell et al., 1984; Shadel
and Clayton, 1997). Indeed there is not normally much oppor-
tunity for mtDNA molecules to meet as they are sequestered
within the double mitochondrial membrane system. Under
certain circumstances mitochondria may have the capacity to
fuse and form stable heteroplasmic lineages (Jenuth et al., 1996;
Jenuth et al., 1997; Meirelles and Smith, 1997, 1998) Indeed
mitochondrial fusion as well as fission is normal in oogenesis
and embryogenesis (Smith and Alcivar, 1993). There is also
a complex tissue-specific and species-specific set of interactions
between the nuclear and mitochondrial genomes that govern
both transcription and mtDNA replication (Poyton and McEwen,
1996). It is possible that the lack of mitochondrial HR is a
fundamental aspect of eukaryotic evolution that is possibly
based on the need to provide the next generation with a
highly competent OXPHOS capacity. The generation of di-
versity by mitochondrial recombination could lead to lethal
genomic conflicts and hence reduced fitness (Hurst, 1992). To
coin an analogy: if sex is democracy and mitochondria are
the slaves of the organism, then democracy for the slaves is a
luxury the organism can well do without.
Conclusions
The role of mitochondria in the reproductive life cycle still
holds many mysteries. Key questions remain to be answered.
What is the mechanism that recognizes and eliminates same-
species but not foreign spermatozoa in the early embryo?
Do sperm mitochondria have functional mitochondrial
import and export mechanisms? If so can they respond to
transcription factors in vitro, in the embryo and in other cell
systems? Can they evade the normal surveillance mechanisms
of the embryo?
Is the location of the sperm midpiece after syngamy constant
in relation to embryonic axes?
What relationship is there between mitochondrial integrity
and oocyte atresia?
These ideas could not have been generated without the generosity
of a number of colleagues over the past few years, especially Norman
Hecht, Anne Jequier, Roger Martin, Roger Short, Eric Shoubridge,
Lawrence Smith, Bayard Storey, Peter Sutovsky and Ryuzo Yanagimachi.
Thanks also to Tim Karr and Scott Pitnick for allowing me to quote
from their paper in print.
References
Key references are indicated by asterisks.
Adams CE (1984) Reproductive senescence. In Reproduction in Mammals. Book 4.
Reproductive Fitness (2nd Edn) pp 210–233 Eds CR Austin, RV Short.
Cambridge University Press, Cambridge
Ahmadi A and Ng SC (1997) Sperm head decondensation, pronuclear for-
mation, cleavage and embryonic development following intracytoplasmic
injection of mitochondria-damaged sperm in mammals Zygote 5 247–253
Aitken RJ (1995) Free radicals, lipid peroxidation and sperm function
Reproduction, Fertility and Development 7 659–668
Alcivar AA, Hake LE, Millette CF, Trasler JM and Hecht NB (1989)
Mitochondrial gene expression in male germ cells of the mouse
Developmental Biology 135 263–271
Anderson S, Bankier AT, Barrell BG et al. (1981) Sequence and organization
of the human mitochondrial genome Nature 290 457–465
*Ankel-Simons F and Cummins JM (1996) Misconceptions about mito-
chondria and mammalian fertilization –implications for theories on
human evolution Proceedings of the National Academy of Sciences USA 93
13 859–13 863
Anson RM, Croteau DL, Stierum RH, Filburn C, Parsell R and Bohr VA
(1998) Homogenous repair of singlet oxygen-induced DNA damage in
differentially transcribed regions and strands of human mitochondrial
DNA Nucleic Acids Research 26 662–668
Antczak M and Van Blerkom J (1997) Oocyte influences on early develop-
ment: the regulatory proteins leptin and STAT3 are polarized in mouse
and human oocytes and differentially distributed within the cells of the
preimplantation stage embryo Molecular Human Reproduction 3 1067–1086
Ashley MV, Laipis PJ and Hauswirth WW (1989) Rapid segregation of
heteroplasmic bovine mitochondria Nucleic Acids Research 17 7325–7331
Baker TG (1982) Oogenesis and ovulation. In Reproduction in Mammals. Book 1.
Germ Cells and Fertilization (2nd Edn) pp 17–45 Eds CR Austin and
RV Short. Cambridge University Press, Cambridge
*Bedford JM and Hoskins DD (1990) The mammalian spermatozoon:
morphology, biochemistry and physiology. In Marshall’s Physiology of
Reproduction (4th Edn pp 379–568 Ed. GE Lamming. Churchill Livingston,
London
*Birky CW (1995) Uniparental inheritance of mitochondrial and chloroplast
genes: mechanisms and evolution Proceedings of the National Academy of
Sciences, USA 92 11 331–11 338
Blok RB, Gook DA, Thorburn DR and Dahl HH (1997) Skewed segregation
of the mtDNA nt 8993 (T–>G) mutation in human oocytes American
Journal of Human Genetics 60 1495–1501
Byskov AG (1982) Primordial germ cells and regulation of meiosis. In Germ
Cells and Fertilization (2nd Edn) pp 1–16 Eds CR Austin and RV Short.
Cambridge University Press, Cambridge
Downloaded from Bioscientifica.com at 06/18/2020 09:26:49PM
via free access
 Mitochondrial DNA in the life cycle
Calarco PG (1995) Polarization of mitochondria in the unfertilized mouse
oocyte Developmental Genetics 16 36–43
Cataldo L, Baig K, Oko R, Mastrangelo MA and Kleene KC (1996)
Developmental expression, intracellular localization, and selenium
content of the cysteine-rich protein associated with the mitochondrial
capsules of mouse sperm Molecular Reproduction and Development 45
320–331
Chen X, Prosser R, Simonetti S, Sadlock J, Jagiello G and Schon EA (1995)
Rearranged mitochondrial genomes are present in human oocytes
American Journal of Human Genetics 57 239–247
Clayton DA (1982) Replication of animal mitochondrial DNA Cell 28 693–705
Clermont Y, Oko R and Hermo L (1993) Cell biology of mammalian
spermiogenesis. In Cell and Molecular Biology of the Testis pp 332–376
Eds C Desjardins and LL Ewing. Oxford University Press, New York,
Oxford
Cohen J, Scott R, Schimmel T, Levron J and Willadsen S (1997) Birth of
infant after transfer of anucleate donor oocyte cytoplasm into recipient
eggs Lancet 350 186–187
Cohen J, Scott R, Alikani M, Schimmel T, Munné S, Levron J, Wu L,
Brenner C, Warner C and Willadsen S (1998) Ooplasmic transfer in
mature human oocytes Molecular Human Reproduction 4 269–280
Cortopassi GA and Wang E (1996) There is substantial agreement among
interspecies estimates of DNA repair activity Mechanisms of Ageing and
Development 91 211–218
Cummins JM, Densley E, Jequier AM, Meloni BP and Sutarno (1994a)
Human male infertility and mitochondrial DNA. In The Seventh
International Symposium on Spermatology Cairns, p 7.5
Cummins JM, Jequier AM and Kan R (1994b) Molecular biology of human
male infertility – links with aging, mitochondrial genetics, and oxidative
stress? Molecular Reproduction and Development 37 345–362
Cummins JM, Wakayama T and Yanagimachi R (1997) Fate of microinjected
sperm components in the mouse oocyte and embryo Zygote 5 301–308
Cummins JM, Jequier AM, Martin R, Mehmet D and Goldblatt J (1998)
Semen levels of mitochondrial DNA deletions in men attending an in-
fertility clinic do not correlate with phenotype Journal of Andrology 21
47–52
Davis AF and Clayton DA (1996) In situ localization of mitochondrial DNA
replication in intact mammalian cells Journal of Cell Biology 135 883–893
Dawkins R (1986) The Blind Watchmaker (1st Edn) p 176 W.W. Norton and
Company, New York, London
Dawkins R (1995) River out of Eden (1st Edn) Phoenix, London
Ebert KM, Liem H and Hecht NB (1988) Mitochondrial DNA in the mouse
preimplantation embryo Journal of Reproduction and Fertility 82 145–149
*Edwards RG and Beard HK (1997) Oocyte polarity and cell determination in
early mammalian embryos Molecular Human Reproduction 3 863–905
Fawcett DW (1981) The Cell. An Atlas of Fine Structure (2nd Edn) W. B. Saunders
Co, Philadelphia.
Finn CA (1998) Menstruation: a nonadaptive consequence of uterine evol-
ution. Quarterly Review of Biology 73 163–173
Fisher RP, Lisowsky T, Parisi MA and Clayton DA (1992) DNA wrapping
and bending by a mitochondrial high mobility group-like transcriptional
activator protein Journal of Biological Chemistry 267 3358–3367
*Ford WCL and Rees JM (1990) The bioenergetics of mammalian sperm
motility. In Controls of Sperm Motility: Biological and Clinical Aspects
pp 175–202 Ed. C Gagnon. CRC Press, Boca Raton
Garner DL, Thomas CA, Joerg HW, Dejarnette JM and Marshall CE (1997)
Fluorometric assessments of mitochondrial function and viability in cryo-
preserved bovine spermatozoa Biology of Reproduction 57 1401–1406
Gartner K, Bondioli K, Hill K and Rapp K (1998) High variability of body
sizes within nucleus-transfer-clones of calves – artifacts or a biological
feature Reproduction in Domestic Animals 33 67–75
Goldberg E, Sberna D, Wheat TE, Urbanski GJ and Margoliash E (1977)
Cytochrome C: immunofluorescent localization of the testis-specific form
Science 196 1010–1012
Gyllensten U, Wharton D, Josefsson A and Wilson AC (1991) Paternal in-
heritance of mitochondrial DNA in mice Nature 352 255–257
Hayakawa M, Katsumata K, Yoneda M, Tanaka M, Sugiyama S and
Ozawa T (1996) Age-related extensive fragmentation of mitochondrial
DNA into minicircles Biochemical and Biophysical Research Communications
226 369–377
Hayashi J, Takemitsu M, Goto Y and Nonaka I (1994) Human mitochondria
and mitochondrial genome function as a single dynamic cellular unit
Journal of Cell Biology 125 43–50
181
Hecht N (1995) The making of a spermatozoon: a molecular perspective
Developmental Genetics 16 95–103
Hecht N (1997) Genetic control of spermatogenesis: where can things go
wrong? In Genetics of Human Male Fertility pp 11–24 Eds C Barratt, C De
Jonge, D Mortimer and J Parinaud. Editions E.D.K., Paris
Hecht NB and Liem H (1984) Mitochondrial DNA is synthesized during
meiosis and spermiogenesis in the mouse Experimental Cell Research 154
293–298
Hecht NB, Liem H, Kleene KC, Distel RJ and Ho SM (1984) Maternal in-
heritance of the mouse mitochondrial genome is not mediated by a loss or
gross alteration of the paternal mitochondrial DNA or by methylation of
the oocyte mitochondrial DNA Developmental Biology 102 452–461
Hiraoka J and Hirao Y (1988) Fate of sperm tail components after incor-
poration into the hamster egg Gamete Research 19 369–380
Hogan B, Constantini F and Lacy E (1986) Manipulating the Mouse Embryo.
A Laboratory Manual (1st Edn) Cold Spring Harbor Laboratory, Cold
Spring Harbour
*Holliday R (1995) Understanding Ageing (1st Edn) Cambridge University
Press, Cambridge
Howell N (1997) mtDNA recombination: what do in vitro data mean?
American Journal of Human Genetics 61 19–22
Howell N, Huang P and Kolodner RD (1984) Origin, transmission, and
segregation of mitochondrial DNA dimers in mouse hybrid and cybrid
cell lines Somatic and Cellular Molecular Genetics 10 259–274
Hurst LD (1992) Intragenomic conflict as an evolutionary force Philosophical
Transactions of the Royal Society of London Series B: Biological Sciences 248
135–140
Hurst LD (1995) Selfish genetic elements and their role in evolution – the
evolution of sex and some of what that entails Philosophical Transactions of
the Royal Society of London – Series B: Biological Sciences 349 321–332
*Hurst LD, Atlan A and Bengtsson BO (1996) Genetic conflicts Quarterly
Review of Biology 71 317–364
Jenuth JP, Peterson AC, Fu K and Shoubridge EA (1996) Random genetic
drift in the female germline explains the rapid segregation of mammalian
mitochondrial DNA Nature Genetics 14 146–151
Jenuth JP, Peterson AC and Shoubridge EA (1997) Tissue-specific selection for
different mtDNA genotypes in heteroplasmic mice Nature Genetics 16 93–95
Johns DR (1995) Mitochondrial DNA and disease New England Journal of
Medicine 333 638–644
Kanazawa M, Yano M, Namchai C, Yamamoto S, Ohtake A, Takayanagi M,
Mori M and Niimi H (1997) Visualization of mitochondria with green
fluorescent protein in cultured fibroblasts from patients with mitochondrial
diseases Biochemical and Biophysical Research Communications 239 580–584
Kaneda H, Hayashi JI, Takahama S, Taya C, Lindahl KF and Yonekawa H
(1995) Elimination of paternal mitochondrial DNA in intraspecific crosses
during early mouse embryogenesis Proceedings of the National Academy of
Sciences USA 92 4542–4546
Keefe DL, Niven-Fairchild T, Powell S and Buradagunta S (1995)
Mitochondrial deoxyribonucleic acid deletions in oocytes and repro-
ductive aging in women Fertility and Sterility 64 577–583
Kenyon L and Moraes CT (1997) Expanding the functional human mito-
chondrial DNA database by the establishment of primate xeno-
mitochondrial cybrids Proceedings of the National Academy of Sciences USA
94 9131–9135
King MP and Attardi G (1989) Human cells lacking mtDNA: repopulation
with exogenous mitochondria by complementation Science 246 500–503
Kitagawa T, Suganuma N, Nawa A, Kikkawa F, Tanaka M, Ozawa T and
Tomoda Y (1993) Rapid accumulation of deleted mitochondrial deoxy-
ribonucleic acid in postmenopausal ovaries Biology of Reproduction 49
730–736
Larsson NG, Oldfors A, Holme E and Clayton DA (1994) Low levels of mito-
chondrial transcription factor A in mitochondrial DNA depletion
Biochemical and Biophysical Research Communications 200 1374–1381
Larsson NG, Garman JD, Oldfors A, Barsh GS and Clayton DA (1996)
A single mouse gene encodes the mitochondrial transcription factor A
and a testis-specific nuclear hmg-box protein Nature Genetics 13 296–302
Larsson NG, Oldfors A, Garman JD, Barsh GS and Clayton DA (1997)
Down-regulation of mitochondrial transcription factor A during sper-
matogenesis in humans Human Molecular Genetics 6 185–191
Larsson NG, Wang J, Wilhelmsson H, Oldfors A, Rustin P, Lewandoski M,
Barsh GS and Clayton DA (1998) Mitochondrial transcription factor A is
necessary for mtDNA maintenance and embryogenesis in mice Nature
Genetics 18 231–236
Downloaded from Bioscientifica.com at 06/18/2020 09:26:49PM
via free access
182 J. Cummins
*Lodish H, Baltimore D, Berk A, Zipursky SL, Matsudaira P and Darnell J
(1995) Molecular Cell Biology (3rd Edn) W.H. Freeman and Company,
New York
Machado de Domenech E, Domenech CE, Aoki A and Blanco A (1972)
Association of the testicular lactate dehydrogenase isozyme with a special
type of mitochondria Biology of Reproduction 6 136–147
Manfredi G, Thyagarajan D, Papadopoulou LC, Pallotti F and Schon EA
(1997) The fate of human sperm-derived mtDNA in somatic cells American
Journal of Human Genetics 61 953–960
Marchington DR, Hartshorne GM, Barlow D and Poulton J (1997)
Homopolymeric tract heteroplasmy in mtDNA from tissues and single
oocytes – support for a genetic bottleneck American Journal of Human
Genetics 60 408–416
Max B (1992) This and that: hair pigments, the hypoxic basis of life and the
Virgilian journey of the spermatozoon Trends in Pharmacological Science 13
272–276
Meinhardt A, Parvinen M, Bacher M, Aumuller G, Hakovirta H, Yagi A and
Seitz J (1995) Expression of mitochondrial heat shock protein 60 in dis-
tinct cell types and defined stages of rat seminiferous epithelium Biology of
Reproduction 52 798–807
Meirelles FV and Smith LC (1997) Mitochondrial genotype segregation in a
mouse heteroplasmic lineage produced by embryonic karyoplast trans-
plantation Genetics 145 445–451
Meirelles F and Smith LC (1998) Mitochondrial genotype segregation during
preimplantation development in mouse heteroplasmic embryos Genetics
148 877–883
Müller-Höcker J, Schäfer S, Weis S, Münscher C and Strowitzki T (1996)
Morphological–cytological and molecular genetic analysis of mito-
chondria in isolated human oocytes in the reproductive age Molecular
Human Reproduction 2 951–958
Murphy MP (1997) Selective targeting of bioactive compounds to mito-
chondria Trends in Biotechnology 15 326–330
Nagao Y, Totsuka Y, Atomi Y, Kaneda H, Lindahl KF, Imai H and
Yonekawa H (1998) Decreased physical performance of congenic mice
with mismatch between the nuclear and the mitochondrial genome Genes
and Genetic Systems 73 21–27
Nagley P, Zhang C, Martinus RD, Vaillant F and Linnane AW (1993)
Mitochondrial DNA mutation and human aging: molecular biology, bio-
energetics, and Redox therapy. In Mitochondrial DNA in Human Pathology
pp 137–157 Eds S DiMauro and D Wallace. Raven Press, New York
Neupert W (1997) Protein import into mitochondria Annual Review of
Biochemistry 66 863–917
Nosek J, Tomaska L, Fukuhara H, Suyama Y and Kovac L (1998) Linear
mitochondrial genomes – 30 years down the line Trends in Genetics 14
184–188
Olivo PD, Van de Walle MJ, Laipis PJ and Hauswirth WW (1983)
Nucleotide sequence evidence for rapid genotypic shifts in the bovine
mitochondrial DNA D-loop Nature 306 400–402
Olson GE and Winfrey VP (1992) Structural organization of surface domains
of sperm mitochondria Molecular Reproduction and Development 33 89–98
Otani H, Tanaka O, Kasai K and Yoshioka T (1988) Development of mito-
chondrial helical sheath in the middle piece of the mouse spermatid tail:
regular dispositions and synchronized changes Anatomical Record 222
26–33
*Ozawa T (1997a) Genetic and functional changes in mitochondria associated
with aging Physiological Reviews 77 425–464
Ozawa T (1997b) Oxidative damage and fragmentation of mitochondrial
DNA in cellular apoptosis Bioscience Reports 17 237–250
Pikó L and Matsumoto L (1976) Number of mitochondria and some proper-
ties of mitochondrial DNA in the mouse egg Developmental Biology 49 1–10
Pikó L and Taylor KD (1987) Amounts of mitochondrial DNA and abun-
dance of some mitochondrial gene transcripts in early mouse embryos
Developmental Biology 123 364–374
Pinkert CA, Irwin MH, Johnson LW and Moffatt RJ (1997) Mitochondria
transfer into mouse ova by microinjection Transgenic Research 6 379–383
Pitnick S and Karr TL (1998) Paternal products and by-products in Drosophila
development Proceedings of the Royal Society of London, Series B 265 1–6
Poyton RO and McEwen JE (1996) Crosstalk between nuclear and mitochon-
drial genomes Annual Review of Biochemistry 65 563–607
Rinaudo P, Niven-Fairchild T, Buradugunta S, Massabrio M and Keefe D
(1996) Preimplantation embryo development does not screen out mutant
mitochondrial DNA (mtDNA). In American Society for Reproductive
Medicine Annual Conference Boston pp P-015
Roveri A, Maiorino M, Nisii C and Ursini F (1994) Purification and charac-
terization of phospholipid hydroperoxide glutathione peroxidase from rat
testis mitochondrial membranes Biochimica et Biophysica Acta 1208 211–221
Salazar JJ and Vanhouten B (1997) Preferential mitochondrial DNA injury
caused by glucose oxidase as a steady generator of hydrogen peroxide in
human fibroblasts Mutation Research – DNA Repair 385 139–149
Schultz RA, Swoap SJ, McDaniel LD, Zhang BQ, Koon EC, Garry DJ, Li K
and Williams RS (1998) Differential expression of mitochondrial DNA
replication factors in mammalian tissues Journal of Biological Chemistry 273
3447–3451
Seitz J, Mobius J, Bergmann M and Meinhardt A (1995) Mitochondrial dif-
ferentiation during meiosis of mole germ cells International Journal of
Andrology 18 7–11
*Shadel GS and Clayton DA (1997) Mitochondrial DNA maintenance in ver-
tebrates Annual Review of Biochemistry 66 409–435
Shitara H, Hayashi J, Takahama S, Kaneda H and Yonekawa H (1998)
Maternal inheritance of mouse mtDNA in interspecific hybrids – segre-
gation of the leaked paternal mtDNA followed by the prevention of
subsequent paternal leakage Genetics 148 851–857
Shoffner JM and Wallace DC (1990) Oxidative phosphorylation diseases.
Disorders of two genomes Advances in Human Genetics 19 267–330
Short RV (1998) The difference between a testis and an ovary Journal of
Experimental Zoology 281 (in press)
Smith LC and Alcivar AA (1993) Cytoplasmic inheritance and its effects on
development and performance Journal of Reproduction and Fertility
Supplement 48 31–43
Steinborn R, Zakhartchenko V, Jelyazkov J, Klein D, Wolf E, Muller M and
Brem G (1998a) Composition of parental mitochondrial DNA in cloned
bovine embryos FEBS Letters 426 352–356
Steinborn R, Zakhartchenko V, Wolf E, Muller M and Brem G (1998b)
Non-balanced mix of mitochondrial DNA in cloned cattle produced by
cytoplast-blastomere fusion FEBS Letters 426 357–361
*Sutovsky P, Navara CS and Schatten G (1996) Fate of the sperm mito-
chondria, and the incorporation, conversion, and disassembly of the
sperm tail structures during bovine fertilization Biology of Reproduction 55
1195–1205
Tarin JJ (1996) Potential effects of age-associated oxidative stress on mam-
malian oocytes/embryos Molecular Human Reproduction 2 717–724
Taylor RW, Chinnery PF, Clark KM, Lightowlers RN and Turnbull DM
(1997) Treatment of mitochondrial disease Journal of Bioenergetics and
Biomembranes 29 195–205
Thyagarajan B, Padua RA and Campbell C (1996) Mammalian mitochondria
possess homologous DNA recombination activity Journal of Biological
Chemistry 271 27 536–27 543
Van Blerkom J (1997) Can the developmental competence of early human
embryos be predicted effectively in the clinical IVF laboratory? Human
Reproduction 12 1610–1614
Van Blerkom J, Davis PW and Lee J (1995) ATP content of human oocytes
and developmental potential and outcome after in vitro fertilization and
embryo transfer Human Reproduction 10 415–424
*Wallace DC (1997) Mitochondrial DNA in aging and disease Scientific
American 277 40–47
Willoughby CE, Mazur P, Peter AT and Critser JK (1996) Osmotic tolerance
limits and properties of murine spermatozoa Biology of Reproduction 55
715–727
Wilmut I, Schnieke AE, McWhir J, Kind AJ and Campbell KHS (1997) Viable
offspring derived from fetal and adult mammalian cells Nature 385 810–813
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