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Reviews of (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 in eukaryotic . 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 – imposes different evolutionary constraints on males and , and may lie at the heart of . This review examines the implications of recent findings on the of mtDNA for reproduction and inheritance in mammals.

Although the existence of mitochondria has been known since encodes for tryptophan in mammalian mitochondria). The in- the last century, mitochondrial DNA (mtDNA) has been studied heritance of mitochondria through the female lineage remains most extensively in the past two decades. Mitochondria have one of the central enigmas of reproductive biology. a profound role to play in mammalian tissue bioenergetics, in Mitochondria have certain tissue-specific configurations that growth, in ageing and in , and yet they descend from presumably reflect local energetic requirements (Fawcett, 1981). an asexually reproducing independent life form. It has been New techniques for visualizing mitochondria in whole cells, hypothesized that tensions between the evolutionary ‘interests’ such as the incorporation of green fluorescent coupled of the eukaryotic host and its subservient organelles have led to with confocal microscopy, reveal that mitochondrial form can asymmetrical inheritance, so that mitochondria derive pre- not only reflect pathological states but may also be extremely dominantly from the female in most organisms (Hurst, 1992; diverse even within the same (Kanazawa et al., 1997). The Hurst et al., 1996). This has consequences that are only just be- basic design is a double membrane surrounding an inner mito- coming apparent. There is considerable interest in the potential chondrial matrix that contains one or more circular mtDNA role of mitochondria and cytoplasmic inheritance on growth molecules. The outer mitochondrial membrane is thought to and performance factors such as muscle development and milk represent the original invaginated host plasma membrane, production in domestic . As might be expected, the while the inner mitochondrial membrane represents the bac- maternally inherited mitochondrial genome has significant terial wall and contains the site of oxidative phosphorylation non-Mendelian effects on steroidogenesis and on respiratory- (OXPHOS) on its inner surface. The matrix also contains ribo- dependent functions such as growth, oxygen consumption and somes for local protein synthesis. lean:fat ratios, but not on anaerobic metabolism. Smith and The two membranes differ profoundly in composition. The Alcivar (1993) have reviewed this topic recently and compre- lipid:protein ratio of the outer membrane is about 50:50 and hensively; therefore, the focus of this article will be the role of it is permeable to molecules with molecular weights of up to mtDNA in the life cycle. 10 000 (Lodish et al., 1995). The inner membrane is relatively Mitochondria are semi-autonomous organelles found in all impermeable and is about 80% protein and is thrown up into eukaryotic cells (except mature red blood cells and some pro- infoldings – crystae – the sites of OXPHOS enzymes. Here the tozoans). It is generally accepted that mitochondria originated oxidation of metabolites generates ATP through a series of in ancestral eukaryotic cells through endosymbiosis of free- integral membrane multi-subunit protein complexes which living capable of metabolizing oxygen – a suggestion couple electron transport to ATP synthesis. These complexes first made over a century ago and noted by Ozawa in his review are unique in that they consist of encoded by two (Ozawa, 1997a). Our ancestral thus exploited the separate yet cooperating , that of the nucleus and that capacity of mitochondria to metabolise oxygen. This allowed of the mitochondrion (Poyton and McEwen, 1996; Shadel and them to flourish despite the increasing concentrations of this Clayton, 1997) There are separate translocase systems in the highly reactive and potentially poisonous element in the en- inner and outer membranes that coordinate the recognition, vironment. While most of the mitochondrial have moved import and assortment of essential proteins from the cytosol to the nucleus, mitochondria retain their bacterial facility for (Neupert, 1997). multiplying by simple fission – and even fusing – indepen- dently of the host . Excess mitochondria are removed Mitochondrial DNA by autophagic lysosomal activity. These population control measures act in response to the energetic demands of different Most cells in the body contain between 103 and 104 copies of tissues. The mitochondrial genome also has idiosyncrasies in mtDNA. There are much higher copy numbers (about 105) in RNA processing and in its genetic code that differ from those of mature . This may be in preparation for the energetic nuclear DNA (for example UGA is normally a stop codon, but demands of embryogenesis (Pikó and Matsumoto, 1976) but © 1998 Journals of Reproduction and 1359-6004/98 $12.50 Downloaded from Bioscientifica.com at 09/24/2021 01:14:10PM via free access Mitochondrial DNA in the life cycle 173

Table 1. Fate of light and heavy chain transcripts from mitochondrial DNA Displaced heavy strand (D-loop) Light chain transcripts Heavy chain transcripts Heavy strand Nascent heavy origin strand 8 tRNAs 14 tRNAs 1 mRNA 12 mRNAS Heavy strand RNA primers for heavy chain replication 2 tRNAs promoter Modified from Shadel and Clayton, 1997 factor binding Light strand sites promoter 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 Light strand origin exists mainly as a circular molecule of approximately 16.6 kb, (stem-loop) 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 Fig. 1. This diagram summarizes the major features of mtDNA re- ferred to in the text. (See Shadel and Clayton, 1997.) 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- Box 1 Major mitochondrial import proteins vention mtDNA is depicted as a circle (Fig. 1), but alternatives, such as dimer loops and catenated (chain-linked) circles, are Mitochondrial DNA and RNA polymerases known (Clayton, 1982). In some single-celled organisms (many Transcription, translation and transcription termination factors RNA processing enzymes pathogenic to mammals) aberrant linear mtDNA molecules Mitochondrial ribosomal proteins with -like endings are also found (Nosek et al., 1998). Aminoacyl-tRNA synthetases 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- Neupert, 1997). Besides structural components of the mitochon- ical for the initiation of transcription and translation (see below). dria, these imports involve factors that regulate and specifically The mammalian mitochondrial genome is extremely com- recognize mtDNA and regulate expression (Box 1). pressed with no introns. Some genes even overlap. This con- trasts strikingly with, for example, (five times larger at Nuclear–mitochondrial interactions 78 000 bp) or , in which there are multiple recombining molecules with enormous size variation even within a . Any alterations that arise in the components of the mtDNA For example, sizes vary from 330 000 bp in watermelons to or RNA that recognize or bind to nuclear-encoded regulatory 2.5 + 106 bp in muskmelon (Lodish et al., 1995). Most of the 100 elements must be balanced by compensatory in the or more genes controlling the synthesis of mammalian mito- nuclear genes, as the mitochondrial genome mutates much chondrial proteins have moved to the nucleus over the course more rapidly than the nuclear genome (see below). This mu- of . 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 Import–export mechanisms expression between the nuclear and mitochondrial genomes. Proteins are assembled on cytoplasmic ribosomes and trans- However, it is clearly a sensitive system. Nagao et al. (1998) ported into the mitochondrion through protein-lined channels, found decreased physical performance and growth rates in with the aid of cytoplasmic chaperone proteins (Box 1). These mice in which the nuclear and mitochondrial genes were mis- import mechanisms are prime targets for strategies aimed at matched. The mitochondrial genome exists as multiple mitochondrial genome therapy or modification (Murphy, 1997; copies per cell and yet for unknown reasons proteins encoded Taylor et al., 1997). Import involves hydrolysis of ATP in both the by single copy nuclear genes are present in the same amounts cytoplasm and the mitochondrion plus an electrochemical poten- as mtDNA-encoded proteins (Poyton and McEwen, 1996). This tial across the inner mitochondrial membrane (Lodish et al., 1995; is clearly an issue that will bear on attempts to clone animals,

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Table 2. Summary of some major mtDNA functional locations in the mtDNA. While the ‘common’ 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 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 (O ) is within the D-loop, in tissue bioenergetics and ageing H 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; , 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 . 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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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 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 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 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 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 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

Downloaded from Bioscientifica.com at 09/24/2021 01:14:10PM via free access 176 J. Cummins relative concentration of selenium of any cell line in the body, What happens at fertilization? and animals fed a selenium-deficient diet show midpiece defects (Cataldo et al., 1996). The protein is probably the anti- A flood of misinformation in undergraduate textbooks and oxidant, phospholipid hydroperoxide glutathione peroxidase popular texts is promoting the ‘African Eve’ model of human (Roveri et al., 1994), which is intriguing given the known evolution and minimizes and even denies the possibility of susceptibility of spermatozoa to oxidative damage and lipid paternal mtDNA transmission (Ankel-Simons and Cummins, peroxidation (Aitken, 1995). 1996). At least one well-respected author (Richard Dawkins) Other sperm-specific unique mitochondrial proteins also has claimed (but later retracted) that spermatozoa are too small appear during spermiogenesis. These include a sperm-specific to contain mitochondria, a statement that comes as a surprise to lactic dehydrogenase isoenzyme that appears early (Machado most biologists (Dawkins, 1986, 1995). In fact, total sperm entry de Domenech et al., 1972) and a unique isoform of cytochrome c into the including the midpiece mitochondria is the rule (Goldberg et al., 1977). The nuclei lose the mito- rather than the exception for most mammals. However, it is chondrial targeting sequence of a testis-specific isoform of nu- now well established that the mitochondria from spermatozoa clear mitochondrial transcription factor A (Larsson et al., 1994, are targeted for destruction by endogenous proteolytic activity 1997), suggesting that there is no need for mitochondria to during early embryogenesis (see below). Uniparental (gener- replicate once on the midpiece. However, the mitochondrial ally but not universally maternal) inheritance of cytoplasmic transcriptional potential of the is apparently un- organelles like mitochondria and, in plants, plastids, is ac- changed during this process (Alcivar et al., 1989). Larsson et al. complished by a wide variety of strategies and thus is clearly of (1996) suggest that the nuclear isoform of mtTFA, which is also profound importance to long-term fitness. It is usually argued present in , plays a secondary structural role in that uniparental inheritance in sexually reproducing organisms compacting nuclear DNA during spermiogenesis. This would minimizes the risk of lethal genomic conflict between asexually be consistent with its known capacity to induce conformational reproducing cytoplasmic genes (Hurst, 1992, 1995; Hurst et al., changes in DNA (Fisher et al., 1992). 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- Role of mitochondria in sperm function anisms found in different organisms is matched only by the Although the mitochondrial sheath of spermatozoa clearly variety of hypotheses devised to explain the evolution of has a functional role, rather surprisingly there is little consen- the phenomenon”. Perhaps the most bizarre example is in the sus about the relative importance of mitochondrial respiration giant spermatozoa of some species of Drosophila, in which (as opposed to glycolysis) for motility, or even for fertilization the male mitochondria are sequestered to the midgut during itself (Bedford and Hoskins, 1990). Spermatozoa in which the organogenesis and defaecated by the larvae soon after hatching mitochondria have been poisoned with cyanide can induce (Pitnick and Karr, 1998). normal embryo development when microinjected into oocytes (Ahmadi and Ng, 1997). Spermatozoa are metabolically flexible Paternal transmission – an anomaly? and, in some species, can switch between aerobic and anaerobic metabolism. This perhaps reflects the great range of oxygen Most of the evidence indicating the possibility of paternal tensions they experience, from near anoxia in the testis and transmission of mtDNA derives from interspecific crosses, epididymis to ambient tensions in the vagina and in vitro which by definition are uncommon in nature (Kaneda et al., (Ford and Rees, 1990; Max, 1992; Cummins et al., 1994b). New 1995). Backcrossing studies in Mus spretus + Mus musculus techniques, such as flow cytometry using vital dyes and fluoro- hybrids showed a ‘leakage’ of paternal mtDNA of about 1 in chromes, that can indicate the degree of mitochondrial mem- 1000 to 1 in 10 000 molecules (Gyllensten et al., 1991). This is not brane potential promise to elucidate the relationship between far from the theoretical ratio of to sperm mtDNA seen at mitochondrial function, overall sperm viability and fertilizing fertilization (Ankel-Simons and Cummins, 1996) but this obser- potential (Garner et al., 1997). Like somatic mtDNA, that of vation of leakage may be an artefact (see below). Kaneda et al. spermatozoa is highly vulnerable to mutation, and a significant (1995) investigated this question in an ingenious manner, using number of mtDNA deletions are found in the semen of at least a nested polymerase chain reaction (PCR) assay that could de- 50% of normospermic men (Cummins et al., 1998). Given the tect sperm mtDNA in a single mouse embryo. In embryos pro- lengthy process of spermiogenesis and epididymal maturation duced by in vitro fertilization of Mus musculus with the during which the sperm mitochondria have to survive without same strain of spermatozoa, paternal mtDNA could be detected any interaction with the nuclear genome, and given the likeli- in but only at the early pronuclear stage, and its dis- hood that they will be exposed to mutagenic agents, this is per- appearance coincided with loss of membrane potential in haps not surprising. Indeed, the need to exclude defective sperm-derived mitochondria, as measured by Rhodamine 123 sperm mtDNA from contributing to the embryo is possibly one fluorescence. By contrast, when Mus spretus spermatozoa were of the major selection pressures against survival of paternal used to fertilize M. musculus eggs, paternal mtDNA was de- mtDNA. Indeed Short (1998) has suggested that this asym- tected at the pronucleus stage, at the two-cell stage and in metric inheritance of mtDNA, through the oocyte but not the neonates. This was confirmed by sequencing the PCR product. spermatozoon, may be a fundamental driving force behind When spermatozoa from the congenic strain b6.mt(spr), which amphimixis and anisogamy. This is because of the need to con- carries M. spretus mtDNA on a background of M. musculus serve a healthy stock of mtDNA for embryo development, nuclear genes, was used, the M. Spretus mtDNA was elimi- through a long period of quiescence in meiosis. nated early in development, as in intraspecific crosses (Kaneda

Downloaded from Bioscientifica.com at 09/24/2021 01:14:10PM via free access Mitochondrial DNA in the life cycle 177 et al.,1995). Kaneda et al. (1995) proposed that the cyto- Table 3. Possible mechanisms for uniparental inheritance of mammalian plasm has a species-specific mechanism that recognizes and mitochondria eliminates sperm mitochondria, on the basis of nuclear DNA encoded proteins in the sperm midpiece, and not on the Mechanism Evidence in mammals? mtDNA itself, nor on the proteins it encodes. In further work, the same group found that the ‘leaked’ paternal mtDNA fol- Prezygotic lowing interspecific crosses is limited to the first generation, Anisogamy – disparity in numbers between Yes does not get transmitted to all tissues and does not survive eggs and spermatozoa into subsequent back-crosses (Shitara et al., 1998). This calls into Exclusion of mitochondria from spermatozoa No question the original findings of Gyllensten et al. (1991) and Degradation of mitochondria in spermatozoa No suggests that the congenic mouse strain they used in detecting Degradation of mtDNA in spermatozoa Probable paternal leakage may have retained a stable heteroplasmy. This Fertilization may have arisen from mitochondrial fusion as is thought to Exclusion of sperm mitochondria from zygote Very rare have occurred in at least one line of mice produced by cytoplast transfer (Meirelles and Smith, 1997). Zygotic/deterministic Although this picture appears convincing, there are some Selective degradation of sperm mitochondria Yes problems with these data and the results have not been repli- Selective degradation of sperm mtDNA No cated by other laboratories. First, nested PCR detection of very Targeted partitioning of sperm mitochondria Remotely possible small amounts of DNA is fraught with problems of potential into non-embryonic cells contamination, and most laboratories insist on dual facilities Zygotic/stochastic with double-blind controls when carrying out procedures such Random partitioning of sperm mitochondria Remotely possible as embryo sexing. It is not clear whether such precautions into non-embryonic cells 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- embryos have a generalized defence as opposed to an intra- vations using the stable fluorochrome MitoTrackerr (Cummins specific defence against exogenous mitochondria. Hecht (1984) et al., 1997) in which viable midpiece mitochondria were found was unable to detect microinjected conspecific testicular and up to day 3 in four-cell embryos and up to day 5 in non- liver mitochondria in offspring but, more recently, transmission activated oocytes and arrested two- and four-cell embryos. of mutant human mtDNA (from an individual who died of By contrast, other sperm tail components, such as the coarse mitochondrial disease) has been reported in mouse embryos tail fibres, can be identified up to the blastocyst stage and poss- (Rinaudo et al., 1996). Pinkert at al. (1997) have also reported that ibly even later. Clearly there is species-specific recognition and Mus spretus liver mitochondria microinjected into Mus domesticus destruction of the mitochondrial sheath, but it occurs later in zygotes persist to the blastocyst stage. Such models will be very embryogenesis than was proposed by Kaneda et al. (1995). valuable tools for the study of mtDNA disease transmission Moreover the mitochondria in the interspecific-cross embryos and in the search for means to target and perhaps even cure of Kaneda and co-workers (in which the paternal mtDNA per- such disorders (Taylor et al., 1997). sisted) should have been visible by light microscopy, but the authors made no comment on this. Findings in other rodent How are sperm mitochondria recognized by the zygote? species and cattle (summarized in Cummins et al., 1997) all in- dicate independently that sperm mitochondria persist into the At present the mechanism that recognizes and targets the second cell cycle before lysis. Sutovsky et al. (1996) proposed sperm midpiece for destruction is not known although there that the mitochondrial sheath in cattle may be tagged with is now evidence that it is mediated by ubiquitin (Sutovsky, ubiquitin for proteolytic destruction and they now have pre- personal communication). This is one of the central mysteries liminary evidence confirming this interesting idea (Sutovsky, of reproductive biology. The key may lie in the asynchrony personal communication). 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 Are sperm mitochondria viable? cannot maintain membrane potential up to day 6.5 when repli- Single mitochondria can colonise mtDNA- cation of the mtDNA of the embryo begins (Pikó and Taylor, depleted cell lines under experimental conditions in vitro, albeit 1987; Ebert et al., 1988). Leakage of key components such as at low efficiency (King and Attardi, 1989). Manfredi et al. (1997) cytochrome c, a known trigger for apoptosis (Ozawa, 1997b) found even lower long-term colonization when spermatozoa from the mitochondria, may signal multivesicular bodies to were fused with mitochondrially depleted somatic cell lines surround the mitochondria and digest them (Hiraoka and in vitro. Only 10–20% of cells had mitochondria demonstrating Hirao, 1988) Against this must be set the observations on normal transmembrane potential when examined 4 h after persistence of paternal mtDNA in interspecific mouse crosses fusion. One possibility is that the highly differentiated sperm (Kaneda et al., 1995; Shitara et al., 1998). mitochondria lack functional protein import mechanisms and Several other possibilities are summarized on the basis of therefore cannot respond to nuclear-encoded signals when they Birky (1995) (Table 3). The actual mechanism remains to be receive them (Neupert, 1997). It is not yet clear whether eggs and elucidated fully.

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Embryogenesis, and the The role of leptin in the embryo is obscure but presumably it is involved with allocation between the ICM and the ‘restriction–amplification’ model of mtDNA transmission trophectoderm. Clearly our ideas about the ‘totipotency’ of With very few exceptions such as mussels, most organisms the individual embryo cells up to the eight-cell stage need to appear intolerant of heteroplasmy for mtDNA, even though be revised in a major way (Edwards and Beard, 1997). stable heteroplasmic lineages can be created artificially (Jenuth That there is significant polarity in the oocyte and embryo et al., 1996, 1997). When it does occur it generally arises by may help to explain the skewed segregation of mitochondrial de novo mutation during oogenesis and in humans is frequently genotypes in heteroplasmic mice created by cytoplast or associated with progressive mitochondrial bioenergetic dis- karyoplast fusion. When mitochondria were placed in the eases in offspring. Many homoplasmic mutations are probably periphery of oocytes (cytoplast transplantation) the resulting lethal in early embryogenesis owing to oxidative phosphoryl- mice showed significantly more variation than when mito- ation insufficiency (Ozawa, 1997a; Wallace, 1997). Neutral chondria were placed near the (karyoplast derived) nucleus mutations that arise can become fixed in the very (Meirelles and Smith, 1997, 1998). As it is not possible to place rapidly – within one to three generations (Ashley et al., 1989). cytoplasts accurately to the primordial oocyte axis before One possibility is that this may arise through a restriction– electrofusion there can be less certainty that they will end up in amplification process during the exponential growth phase of the ICM and hence in the somatic (and germ) cells of the oogenesis, in which mitochondria multiply by division from a embryo. Another factor to consider is that mitochondrial repli- few precursors (Marchington et al., 1997; Nagley et al., 1993). cation starts in regions of the cytoplasm close to the nucleus Blok et al. (1997) even suggested that the progenitor pool could (Shadel and Clayton, 1997), so spatial positioning may affect be a single mitochondrion in humans. During oogenesis, this mitochondrial transmission. pool expands considerably resulting in about 105 mitochondria In mice the amount of mtDNA – and presumably therefore in the mature oocyte (Pikó and Taylor, 1987). the number of mitochondria – does not change during develop- Alternatively, Jenuth et al. (1996, 1997) suggest that the ‘bottle- ment up to the blastocyst and early egg cylinder stage (Ebert neck’ may be an artefact caused by random genetic drift during et al., 1988). There are approximately 910 cells at day 6.5 early embryogenesis. By contrast, Steinborn et al. (1998a,b) found (Hogan et al., 1986) when the primordial germ cells (PGCs) that the ratio of different mtDNAs found in cattle, produced by first differentiate from the proximal epiblast, so if mitochondria fusion of nucleated blastomeres with enucleated IIm are randomly apportioned between cells during this time, oocytes, was closely related to the stage of the donor embryo: the founder PGC will receive approximately 100 founder 13–18% when the donor was a 24-cell morula and 0.4–0.6% when mitochondria. If mitochondria are not equally segregated to the donor was a 92-cell morula. Thus there was no doubt that this cell line, the number could be even smaller (Olivo et al., donor embryo mtDNA can persist in such heteroplasmic clones. 1983; Ashley et al., 1989). This is a similar order of magnitude This hypothesis is currently contentious. An anonymous to the numbers in oogonia proposed by Jenuth et al. (1996) reviewer of the present paper stated “The clonal expansion of (see above). It is possible that only follicles containing oocytes a limited pool of mtDNA precursors could be likened to an with a high degree of homoplasmy are permitted to achieve urban myth: there is no direct evidence for it, and perhaps no . One way this could be tested would be to evaluate need to propose it”. One trusts that the myth, if it is one, will OXPHOS parameters and mtDNA integrity in dominant com- soon be resolved. pared with atretic oocytes.

Mitochondria in oogenesis and axis formation in embryogenesis Mitochondrial DNA in the ageing The mammalian oocyte contains 2 × 105 copies of the mito- Unlike spermatogenesis which is continuous from puberty, chondrial genome in approximately 100 000 mitochondria. oogenesis starts during fetal life (Baker, 1982; Byskov, 1982). In immature oocytes these appear to be evenly distributed; Oogonia proliferate by , reaching a peak in mid- however, Calarco (1995) found that a distinct polarity appeared gestation. In most mammals, oogonia enter meiosis at this time after meiosis re-started. A cortical group of mitochondria but arrest at dictyotene. Oocyte growth resumes with recruit- marks the extrusion point of the first . Other periph- ment of primordial follicles during the ovulatory cycle and eral foci of mitochondria tend to be concentrated in the hemi- meiosis normally completes only at about the time of sphere containing the metaphase II spindle. New observations and fertilization. Thus there is an extended time (50 years or on the distribution of leptin and STAT3 (Antczak and Van more in women) when the nuclear and cytoplasmic integrity of Blerkom, 1997) demonstrate that the axes of the oocyte and the oocyte need to be maintained without . Atresia resulting embryo are determined very early – perhaps by pos- at all stages means that the vast majority of oocytes never make itioning in relation to blood flow and oxygen availability in the it to ovulation: of the 7 million found at mid-gestation in follicle. Leptin is the 16 kDa cytokine product of the obese gene humans only a few hundred will ever reach ovulatory potential (ob) and activates STAT3, one of the Signal Transducer and (Baker, 1982). Lactational amenorrhoea coupled with birth Activation of Transcription family of proteins. The first cell div- spacing of about four years in hunting–gathering societies ision is meridional resulting in two cells with equal leptin/ means that probably only 10–15 oocytes were ever released in a STAT3 gradients. However, the second cell cycle has one woman’s life while humans were evolving. Even the menstrual meridional and one equational division. This results in one cycle may have developed as a non-adaptive consequence of blastomere being leptin/STAT3 deficient and this cell goes uterine evolution (Finn, 1998). Not surprisingly, the fertility on to form the inner cell mass (ICM) of the blastomere (Fig. 2). of individual oocytes declines with age along with reduced

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Fertilization

Two-cell mtDNA transcription starts

Syngamy Polarity established

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

Germ cell line

Tissue differentiation Day 6Ð7 Ð 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 (Tarin, 1996). However, the data will be hard to evaluate until (Adams, 1984). There is considerable interest in the potential accurate methods for quantifying mtDNA deletions and point role of mtDNA in this process, together with the effects of mutations have been developed. Even normal human oocytes altered OXPHOS and cellular antioxidant systems on the cyto- can be shown to contain mtDNA deletions and rearrangements skeleton, fertilization and subsequent embryo development using extensive PCR cycling (Chen et al., 1995; Keefe et al.,

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1995). Kitagawa et al. (1993) found large numbers of deletions Conclusions in women aged 45 and over, and Müller-Hocker et al. (1996) The role of mitochondria in the reproductive life cycle still showed age-related declines in OXPHOS enzyme components, holds many mysteries. Key questions remain to be answered. and alterations in mitochondrial morphometry that could cor- What is the mechanism that recognizes and eliminates same- relate with declining fertility. These alterations could relate to species but not foreign spermatozoa in the early embryo? the reduced ATP content and potential for development in the Do sperm mitochondria have functional mitochondrial poorer quality oocytes and embryos found in older women import and export mechanisms? If so can they respond to (Van Blerkom et al., 1995; Van Blerkom, 1997). This is a rich transcription factors in vitro, in the embryo and in other cell potential area for future research, as we may find that oocyte systems? Can they evade the normal surveillance mechanisms potential is defined by intra-ovarian factors such as blood of the embryo? supply and local oxygen tension. Is the location of the sperm midpiece after syngamy constant There are already reports of clinics attempting to improve in relation to embryonic axes? IVF results by donor cytoplasmic transfer in women with What relationship is there between mitochondrial integrity poor quality oocytes and recurrent implantation failure (Cohen and oocyte atresia? et al., 1997, 1998). This was coupled in at least one case with re- moval of cytoplasmic fragments from embryos, but in this case These ideas could not have been generated without the generosity mid-pregnancy testing revealed that the mtDNA was of the of a number of colleagues over the past few years, especially Norman mother not the donor, so that the benefits of manipulation Hecht, Anne Jequier, Roger Martin, Roger Short, Eric Shoubridge, are unclear. 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. Why don’t mitochondria have ? Sex, or more specifically (HR) be- References tween mtDNA molecules, is assumed not to occur. Even when there is clear evidence of heteroplasmy within the cytoplasm Key references are indicated by asterisks. there is no convincing evidence that HR occurs between popu- Adams CE (1984) Reproductive senescence. In Reproduction in Mammals. Book 4. Reproductive Fitness (2nd Edn) pp 210–233 Eds CR Austin, RV Short. lations (Hayashi et al., 1994). However, under some circum- Cambridge University Press, Cambridge stances mitochondrial DNA may be capable of recombination Ahmadi A and Ng SC (1997) Sperm head decondensation, pronuclear for- and short-term repair (Thyagarajan et al., 1996; Salazar and mation, cleavage and embryonic development following intracytoplasmic Vanhouten, 1997; Anson et al., 1998). 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(1996) may be due to a *Ankel-Simons F and Cummins JM (1996) Misconceptions about mito- mistake in the topoisomerase–resolvase complex that separates chondria and mammalian fertilization –implications for theories on and re-seals daughter mtDNA molecules at replication. This is human evolution Proceedings of the National Academy of Sciences USA 93 an imperfect process that frequently results in concatenated 13 859–13 863 Anson RM, Croteau DL, Stierum RH, Filburn C, Parsell R and Bohr VA oligomers and other configurations (Howell et al., 1984; Shadel (1998) Homogenous repair of singlet oxygen-induced DNA damage in and Clayton, 1997). Indeed there is not normally much oppor- differentially transcribed regions and strands of human mitochondrial tunity for mtDNA molecules to meet as they are sequestered DNA Nucleic Acids Research 26 662–668 within the double mitochondrial membrane system. 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