Mitochondrial DNA

Chapter 34

Mitochondrial DNA Its Transmission from Gametes and Embryos Justin C. St. John Mitochondrial Genetics Group, Centre for Genetic Diseases, Monash Institute of Medical Research, Clayton Vic., Australia

Chapter Outline Introduction 429 The Mitochondrial Genome 429 Oxidative Phosphorylation 430 Mitochondrial DNA Replication 431 Segregation, Transmission, and Inheritance of Mitochondrial DNA 431 The Control of Mitochondrial DNA Replication During Development 432 How do Nuclear Transfer Embryos Control These Processes? 434

INTRODUCTION Mitochondrial DNA (mtDNA) is a separate genome located in the cytoplasm of nearly all eukaryotic cells (Anderson et  al., 1981). Its importance in developmental outcome has often been neglected. However, its transmission and replication are strictly regulated during early development, as they are integral to the viability and health of the offspring (St John, 2012). Failure to regulate these processes during oogenesis, in the embryo, and prior to gastrulation can have deleterious consequences for the fetus and the offspring. The development of several assisted reproductive technologies, such as somatic cell nuclear transfer (SCNT) and cytoplasmic transfer, have led to a greater understanding of the importance of the regulation of mtDNA transmission and replication (St John, 2012; St John et al., 2010). For example, the introduction of “foreign” mtDNA into the recipient oocyte compromises the genetic identity of the resultant offspring and its metabolic capacity to the extent that many of the reported health problems associated with cloning (Cibelli et  al., 2002) and cytoplasmic transfer Principles of Cloning. DOI: http://dx.doi.org/10.1016/B978-0-12-386541-0.00040-0 © 2012 2014 Elsevier Inc. All rights reserved.

Mixing of Mitochondrial DNA Genotypes 434 Why Does the Donor Cell Mitochondrial DNA Persist? 434 Toxicity of Somatic Mitochondrial DNA 435 Is There a Relationship between Donor Cell and Recipient Oocyte Mitochondrial DNA that Affects Somatic Cell Nuclear Transfer Outcome? 436 Conclusion 436 Acknowledgement 436 References 436

(Acton et al., 2007; Barritt et al., 2001) are similar to those of the well-documented mitochondrial disorders that affect children and adults (St John, 2012; St John et  al., 2010). However, the regulation of mtDNA transmission and replication in the cloned embryo is not an insurmountable problem and, once rectified, could offer considerably more applications for the livestock industry and for investigations in biomedical science (Bowles et al., 2007).

THE MITOCHONDRIAL GENOME The mitochondrial genome differs in size both between and within a species. In the mouse, the mitochondrial genome is 16.3 kb in size (Bibb et  al., 1981), while in the human and pig, it is approximately 16.6 kb (Anderson et  al., 1981) and 16.7 kb in size (Ursing and Arnason, 1998), respectively. The mitochondrial genome encodes 13 polypeptides of the electron transport chain (ETC; see Figure 34.1) (Anderson et  al., 1981), which generates the majority of cellular ATP through the biochemical process of oxidative phosphorylation (OXPHOS) (Pfeiffer et  al., 2001). 429

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LSP

OHsite

D-loop

rRN

AI

TB

Complex I (NADH dehydrogenase)

rR

ND

6

CY

2

NA

HSP

ND5

Complex III (Cytochrome e reductase)

ND1

Complex IV (Cytochrome e oxidase) Complex V (ATP synthase)

sit

Transfer RNAs (rRNAs)

4

O

L

ND2

ND

e

Ribosomal RNAs (rRNAs)

4L

ND

XI CO

3

ND CO XII

ATP8

I

ATP6

COXII

FIGURE 34.1  The mammalian mitochondrial genome. Mammalian mtDNA genome encodes 13 polypeptides of the electron transfer chain. ND1, ND2, ND3, ND4, Nd4L, ND5, and ND6 are associated with Complex I, and MT-CYB (CytB) is the only mtDNA-encoded subunit of Complex III. COXI, COXII, COXIII encode proteins of Complex IV, and ATP6 and ATP8 are associated with Complex V. MtDNA also encodes two rRNAs (12 S rRNA and 16 S rRNA) and 22 tRNAs. The displacement loop (D-loop) is the control region, where the H-strand promoter region (HSP), the L-strand promoter region (LSP) and the origin of H-strand replication (OH) are located. A secondary control region located between ND2 and COXI consists of 30 bp. It is the site of origin of L- strand replication (OL).

It also encodes 22 transfer RNAs (tRNAs) and two ribosomal RNAs (rRNAs; see Figure 34.1), which assist in the translation of mitochondrial RNA (mtRNA), and contains one non-coding region, known as the displacement loop (D-loop) (Anderson et  al., 1981; Bibb et  al., 1981). One of the main functions of the D-loop is to act as the control region, since it is the site of interaction for the nuclear-encoded transcription and replication factors that translocate to the mitochondrion to mediate transcription and replication (Clayton, 1992). The D-loop region also contains two hypervariable regions (HV1 and HV2), which have specific identifying sequences that discriminate between maternal lineages (Gill et  al., 1994). The mitochondrial genome not only distinguishes between maternal familial traits but it is also indicative of regional variation and our ancestral origins (Wallace et al., 2003). Some of the specific mtDNA haplotypes that are indicative of regional variation confer positive and negative predispositions to a variety of traits, including adaptability to heat tolerance, disease, growth, physical

performance, and fertility (Ruiz-Pesini et al., 2004). These traits are also prevalent in many species, including large animals (Bowles et  al., 2008; El Shourbagy et  al., 2006; Bruggerhoff et  al., 2002; Sutarno et  al., 2002; Tamassia et al., 2004), and offer the potential to produce cloned offspring with specific genetic traits determined by mtDNA haplotypes, as well as exploiting the chosen chromosomal traits that are to be propagated (Bowles et al., 2007).

OXIDATIVE PHOSPHORYLATION Within cells, there are a number of biochemical pathways that are specifically associated with the generation of ATP. These include glycolysis, which takes places within the cell’s cytoplasm, and β-oxidation, the citric acid cycle, and OXPHOS, which are orchestrated in the mitochondrion. The process of OXPHOS takes place in the ETC and generates 32 molecules of ATP to every two produced by glycolysis (Pfeiffer et  al., 2001). However, unlike any other cellular apparatus, the subunits of the ETC are

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encoded by both the mitochondrial and nuclear genomes. The importance of OXPHOS to cellular and organ function and to the organism’s well-being is demonstrated by the large number of mitochondrial diseases that can arise from genetic defects to either the chromosomal or mtDNA-encoded genes (Wallace, 1999). In terms of the chromosomal genes, these defects are inherited in the classical Mendelian manner. However, the genetic defects in mtDNA are either maternally inherited (Giles et al., 1980) or are acquired in a somatic fashion (Polyak et al., 1998), the latter of which contributes to aging, degenerative disorders, and cancer. To date, over 100 maternally inherited pathological point mutations and large-scale deletions have been identified in the mitochondrial genome (Elliott et  al., 2008; McFarland et  al., 2007; Schaefer et  al., 2008; Schapira, 2012). These are associated with myopathies, neuromuscular disorders, deafness and blindness, cardiomyopathies, and endocrinopathies. MtDNA diseases can be organ- or tissue-type specific or multisystem in nature (McFarland et  al., 2007). This arises as mtDNA is randomly segregated just after the onset of gastrulation (Shoubridge and Wai, 2007), with high ATP-requiring cells, such as those found in neural and muscle tissue, often being affected (Schapira, 2012). This means that those tissues comprising cells that do not require high ATP and are therefore less dependent on OXPHOS, such as vascular and blood-type cells (Rae et al., 2011), are unlikely to be affected. Unlike chromosomal genetics, the onset of mtDNA disease is triggered when the mutant load, namely the ratio of mutant to wild type mtDNA, reaches a threshold that results in the cell being deprived of sufficient ATP generated through OXPHOS. When the majority of cells in a tissue or organ have a high mutant load, this negatively affects tissue or organ function. For example, in myoclonic epilepsy with ragged red fibers (MERRF) syndrome, the affected cells harbor approximately 85% mutant load (Boulet et  al., 1992), while Leber hereditary optic neuropathy (LHON) is indicative of 60% mutant loading (Chinnery et al., 2001).

To date, over 100 maternally inherited pathological point mutations and large-scale deletions have been identified in the mitochondrial genome.

MITOCHONDRIAL DNA REPLICATION The reliance of the mitochondrial genome upon the nuclearencoded transcription and replication factors demonstrates the symbiotic relationship between the mitochondrial genome and the cell. Without the presence of the mitochondrial genome, with multiple copies per mitochondrion, and in thousands of copies in each cell, the cell by itself would

not be able to generate sufficient ATP to survive (Wallace, 1999). In culture, mtDNA-less (ρ0) cells can be supported by metabolic substrates such as uridine (King and Attardi, 1989). Consequently, it is essential that cells possess sufficient copies of the mitochondrial genome in order to maintain their normal functions and maintain homeostasis, and that mtDNA replication is strictly regulated to meet appropriate demands for ATP. Indeed, the generation of ATP through the process of OXPHOS is dependent upon the continual transcription and replication of the mitochondrial genome (Trounce, 2000). MtDNA replication is initiated by mtDNA transcription (Clayton, 1992). The key mtDNA transcription factor is mitochondrial transcription factor A (TFAM), which is supported by mitochondrial transcription factor B1 (TFB1M) and TFB2M (Bonawitz et  al., 2006). Once TFAM has generated the complete mitochondrial transcriptional amplicon, a small fragment from the 5′ to 3′ end is cleaved (Chang DDaC, 1985; Xu BaC, 1996). This fragment is utilized as the primer for mtDNA replication by the mitochondrial-specific DNA polymerase (POLG) (Clayton, 1992). POLG is a haloenzyme that consists of a catalytic subunit, DNA polymerase subunit gamma-1 (PolG-α/PolgA, encoded by POLG) and an accessory subunit, DNA polymerase subunit gamma-2 (PolG-β/PolgB) (Copeland, 2008). PolgA possesses 5′→3′ exonuclease activity to ensure proofreading fidelity, while PolgB’s main function is to stabilize the catalytic enzyme. PolgB is normally present in a 2:1 ratio with PolgA, highlighting its capacity to anchor POLG to the genome. The process of mtDNA replication is further mediated through the mtDNA-specific helicase, Twinkle, and the mitochondrial single-stranded DNA-binding protein (mtSSB) (Copeland, 2008; Kucej and Butow, 2007). Mutations to any of these factors can result in mtDNA depletion-like syndromes, in which insufficient mtDNA is available to support ATP generation through OXPHOS; like genetic defects to the mitochondrial genome, this can be severely debilitating or even lethal (Wallace, 1999). The common diseases associated with mtDNA depletion syndrome include infantile mitochondrial myopathy (Poulton et  al., 1994), familial mtDNA-associated liver disease (Spelbrink et  al., 1998), fatal childhood myopathy (Larsson et  al., 1994), skeletal muscle and mitochondrial encephalomyopathy disorders (Siciliano et  al., 2000) and ocular myopathy, exercise intolerance, and muscle wasting (Siciliano et al., 2000).

SEGREGATION, TRANSMISSION, AND INHERITANCE OF MITOCHONDRIAL DNA The population of mtDNA that is normally transmitted during mammalian development is derived from the mtDNA present in the metaphase II oocyte (St John et al.,

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2010). It is therefore not surprising that, as natural fertilization takes place, sperm mtDNA is targeted for elimination prior to the onset of embryonic genome activation in a species-specific manner (St John, 2012). Two potential mechanisms have been proposed: the first involves ubiquitin-mediated proteolysis by the cytoplasm of the oocyte (Sutovsky et  al., 1999); and the second involves the degradation of the mitochondria-specific nucleoids, which takes place during spermatogenesis and subsequent digestion of sperm mtDNA immediately post-fertilization (Nishimura et  al., 2006). These processes are usually restricted to intra-specific crosses within single strains or breeds of animals (Sutovsky et  al., 1999; Gyllensten et  al., 1991; St John and Schatten, 2004). Nevertheless, in offspring generated by inter-specific crossings, sperm mtDNA can be transmitted, as evidenced in, for example, mice (Gyllensten et  al., 1991), sheep (Zhao et  al., 2004), cattle (Sutovsky et  al., 1999), and monkeys (St John and Schatten, 2004). Interestingly, the maternal inheritance of mtDNA is a phenomenon primarily associated with mammalian species. Lower organisms, such as Drosophila (Kondo et  al., 1990) and marine mussels (Zouros et  al., 1992) inherit both maternal and paternal mtDNA, although it has been demonstrated that paternal mtDNA can have a deleterious effect on the offspring, specifically the male offspring (Innocenti et al., 2011).

The population of mtDNA that is normally transmitted during mammalian development is derived from the mtDNA present in the metaphase II oocyte.

When mutant and wild-type mtDNA coexist, otherwise known as heteroplasmy, the mutant mtDNA can randomly segregate to early gastrulating cells and, as such, the tissues that harbor these mutant molecules are not specifically targeted (Shoubridge and Wai, 2007; Jenuth et  al., 1997). The segregation and transmission of mutant mtDNA is often explained by the “mitochondrial bottleneck hypothesis,” which proposes that considerable variation in mutant mtDNA transmitted to the next generation originates from the small “founder” population of mitochondrial genomes present in the primordial germ cells (Marchington et  al., 1997). This hypothesis arose from observations in Holstein cattle, where it was clearly evident that certain naturally occurring variants were fixed within a few generations in these cattle (Hauswirth and Laipis, 1982). However, the bottleneck has also been described as a “purification” process, whereby all variants are tested in a strict Darwinian manner (Jenuth et al., 1997; Bergstrom and Pritchard, 1998). On the one hand, this ensures that the most severe mutations are segregated and transmitted but, as a result of their severity, the

embryo will fail to develop and thus aborts in utero (Fan et  al., 2008; Stewart et  al., 2008; Wallace, 2007). On the other hand, milder mutations persist in the embryo and fetus because they benefit from maternal–fetal support but are then severely affected post-parturition (St John, 2012). In the context of mitochondrial genetics, the maternal– fetal environment is a poor selector of the fitness of an offspring to survive and aspire to good health once born. Nevertheless, mtDNA transmission and inheritance should not be viewed in the light of one genetic bottleneck that is present at gastrulation. It is most likely that these processes are far more complicated and could involve several mtDNA replication and reduction events that span oogenesis, pre-implantation development, and organogenesis. Each of these events will assist in prescribing the amount of heteroplasmic mtDNA acquired by the offspring.

THE CONTROL OF MITOCHONDRIAL DNA REPLICATION DURING DEVELOPMENT The regulation of mtDNA copy number during development can be divided into four key phases (St John, 2012; St John et  al., 2010). The first phase comprises oogenesis, when the primordial germ cells progress to primary oocytes and then mature into metaphase II oocytes. Oogenesis is triggered by formation of the primordial germ cells. In the mouse, this represents approximately 200 copies of mtDNA (Cree et al., 2008; Wai et al., 2008; Cao et  al., 2007) and it is these copies that are inherited and recycled from one generation to the next (St John, 2012; St John et  al., 2010). As the process of oogenesis takes place, these 200 copies expand to approximately 180,000 copies per mature metaphase II oocyte (see Figure 34.2) (Cree et al., 2008; Wai et al., 2008; Cao et al.,

~200 (units of inheritance PGC

>250,000+ Metaphase II

FIGURE 34.2  MtDNA copy number expansion during oogenesis. MtDNA is randomly segregated to the primordial germ cells (PGCs) just after gastrulation. As the oocyte develops and matures, it increases its copy number from approximately 200 copies in the PGCs to more than 250,000 copies in the mature, metaphase II oocyte. Modified from (St John, 2012).

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2007), representing a 900-fold increase in mtDNA copy number. The mtDNA copy number in metaphase II competent oocytes is similar across a range of large animals, comprising approximately 250,000 copies (May-Panloup et  al., 2005; Spikings et  al., 2007). Several hypotheses have been proposed to account for the large increase in mtDNA copy number prior to fertilization. These include the necessity to have sufficient mitochondrial genomes to support ATP production for growth and development during oogenesis and for the process of fertilization (Van Blerkom, 2004; Reynier et  al., 2001). However, another key argument is that this represents an investment in the future development of the embryo, fetus, and offspring (St John, 2012; St John et  al., 2010). Indeed, mouse embryos that are homozygous knockout for the two key mtDNA replication factors, TFAM (Larsson et  al., 1998) and PolgA (Hance et  al., 2005), possess few copies of mtDNA in their oocytes and abort in utero at E6.5 to 7.5 and E8.5 to 10, respectively. Nevertheless, the mtDNA content can discriminate between two distinct populations of germinal vesicle stage oocytes. These oocytes can be identified by staining with the dye, brilliant cresyl blue (Roca et al., 1998). Competent oocytes do not express the enzyme glucose6-phosphate dehydrogenase and, as a result, cannot reduce brilliant cresyl blue (i.e. they are BCB+). However, incompetent oocytes maintain expression of glucose-6-phosphate dehydrogenase and can reduce brilliant cresyl blue (i.e. BCB−). This simple assay can be performed as oocytes are isolated from ovaries (at 0 h). When compared to BCB− oocytes, porcine BCB+ oocytes have significantly greater mean oocyte complex volumes, establish the metaphase II spindle (at 42 h), and, following fertilization, develop as embryos and have significantly higher numbers of mtDNA copy at 0 h of in vitro maturation (approximately 400,000 compared to <100,000 copies) (El Shourbagy et al., 2006; Spikings et al., 2006). During the initial stages of in vitro maturation, there are significant changes in mtDNA copy number, as determined by in vitro culture experiments. For example, during the first 24 h of in vitro culture, mtDNA copy number decreases significantly in BCB+ oocytes but steady-state levels are resumed by the time the metaphase II spindle is established (Spikings et  al., 2007), and mtDNA replication is not initiated until much later during development. Interestingly, supplementation of BCB- oocytes with pure populations of isolated mitochondria from BCB+ oocytes results in fertilization outcomes similar to those of BCB+ oocytes (El Shourbagy et  al., 2006). These mtDNA replication and reduction events are indicative of nucleocytoplasmic synchrony, which is a critical criterion for oocyte maturation. Furthermore, if “foreign” mtDNA is introduced into the oocyte during maturation, the mtDNA reduction and replication events will result in an uncertain

outcome regarding how much foreign mtDNA is transmitted, as it may be subject to positive or negative selection. The second phase of mtDNA regulation occurs during pre-implantation development. While there is a slight increase in mtDNA copy number following fertilization, copy number is significantly reduced after the twocell stage (Spikings et  al., 2007; McConnell and Petrie, 2004). In cattle, this amounts to embryos possessing up to 30-40% of the original mtDNA content present at fertilization (May-Panloup et  al., 2005), while in pig >70% mtDNA is lost (Spikings et al., 2007). These low levels are maintained through to the blastocyst stage, when mtDNA replication is then initiated. However, mtDNA replication is confined to the trophectoderm (Spikings et  al., 2007; Houghton, 2006), which gives rise to the placenta and is marked by increased ATP production (Houghton, 2006). The inner cell mass cells, which are the precursors of the fetus and the source of founder embryonic stem cells, do not replicate their mtDNA (Spikings et al., 2007). Importantly, in terms of nucleocytoplasmic synchrony, phase 2 of mtDNA replication during development is marked by the lack of expression of the key nuclearencoded mtDNA replication factors, nuclear respiratory factor 1 (NRF-1), TFAM, and POLG (May-Panloup et al., 2005; Spikings et al., 2007). However, these are expressed in the trophectodermal cells to initiate their mtDNA replication. In the third phase of development there is a continued reduction in mtDNA copy number in the cells of the inner cell mass (Cao et al., 2007; Facucho-Oliveira et al., 2007). MtDNA copy number decreases to very low levels, which establishes the mtDNA set point (St John, 2012; St John et al., 2010; Facucho-Oliveira et al., 2007; FacuchoOliveira and St John, 2009; Kelly and St John, 2010; Kelly et  al., 2011). The mtDNA set point is defined by undifferentiated cells, which have the potential to differentiate into any cell type of the body and possess very few copies of mtDNA, and in which mtDNA replication is only initiated in order to replenish mtDNA copy number following cellular division. Establishment of the mtDNA set point ensures that all gastrulating cells of the developing embryo possess between 30 and 300 copies of mtDNA (St John, 2012; Facucho-Oliveira et  al., 2007; Kelly et  al., 2011). This includes primordial germ cells, which in female lineages marks the point of mitochondrial genome recycling to the next generation. This is also when any variants that have persisted to this stage of development are randomly distributed to one or all of the endodermal, mesodermal, and ectodermal layers. Establishment of the mtDNA set point ensures that all gastrulating cells of the developing embryo possess between 30 and 300 copies of mtDNA.

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The fourth phase constitutes organogenesis. At this stage of development, mtDNA copy number increases as cells commit to specific fates and in accordance with their specific requirements for ATP production (St John, 2012; Facucho-Oliveira et  al., 2007; Facucho-Oliveira and St John, 2009; Kelly et  al., 2011). As a consequence, those cells with a high dependency upon ATP, such as muscle cells (Miller et  al., 2003), have high copy numbers of mtDNA, while vascular cells such as endothelial cells have very few copies (Rae et al., 2011). Most interestingly, gametes show the greatest differences, with fertilizable oocytes possessing >250,000 copies of mtDNA (MayPanloup et al., 2005; Spikings et al., 2007), while mature sperm with fertilization potential have fewer than 10 copies (Amaral et al., 2007). Within each of the four phases, there are genetic bottlenecks that restrict transmission of mtDNA to the fetus and offspring. However, they often act as processes of enrichment rather than purification, with mutant molecules exhibiting highly skewed distribution.

HOW DO NUCLEAR TRANSFER EMBRYOS CONTROL THESE PROCESSES? The introduction of sophisticated assisted reproductive technologies (Marchington et al., 1997) has enhanced animal production capabilities and offers biomedical research new avenues to develop experimental models to study, among others, disease. However, these technologies, which include SCNT, have largely ignored the importance of the strict transmission and replication of mtDNA during development. This is especially critical, as the mitochondrial genome is regulated by its nuclear-encoded transcription and replication factors, and the process often relies on the use of unselected, divergent populations of mtDNA, which originate from the somatic cell and the recipient oocyte; these are often incompatible and could thus affect the phenotype of the offspring, as well as embryonic development (St John, 2012; St John et al., 2010; Bowles et al., 2007). This point is best highlighted by fish cloning, where carp nuclei were transferred into enucleated goldfish oocytes. The resultant carp exhibit some morphological traits of the goldfish (Sun et al., 2005). As a consequence of the nature of SCNT, i.e. the transfer of a whole cell into an oocyte with a different mtDNA genotype, the genetic identify of offspring is also compromised, meaning that the resulting “clone” is not a “true genetic clone.”

MIXING OF MITOCHONDRIAL DNA GENOTYPES As nuclear transfer often introduces a whole cell into enucleated recipient oocytes, embryos, fetuses and offspring can inherit their mtDNA from three potential sources:

PART  | VI  SCNT as a Tool to Answer Biological Questions

1. The donor cell only; 2. The recipient oocyte only; or 3. A combination of the donor cell and the recipient oocyte. From the large number of reports following SCNT outcomes, the transmission and inheritance of mtDNA can involve contributions from the recipient oocyte only or a combination of the donor cell and the recipient oocyte [reviewed in St John et al., 2010]. The latter of these outcomes has led to the state of heteroplasmy being redefined to include instances where two different populations of wild-type mtDNA coexist. This contrasts with the pathological situation, which is defined by the coexistence of wild type and mutant mtDNAs. However, just as with segregation at gastrulation following normal fertilization, the contribution of donor cell mtDNA to the embryo or the offspring is not indicative of its starting contribution. Indeed, donor cell mtDNA contribution to the total mtDNA content can range from 0 to 63% in the embryo (Meirelles et al., 2001) and 0 to 59% in the offspring (Takeda et  al., 2003), where the initial mtDNA contribution of the donor cell ranges from approximately 1100 to 4300 copies of mtDNA (Bowles et  al., 2007). Furthermore, the random presence of donor cell mtDNA is not related to whether intra- or inter-specific SCNT has been performed (St John et  al., 2004 Jun). For example, donor mtDNA is present in bovine embryos derived by both intra- (Steinborn et  al., 1998) and inter-specific nuclear transfer (Meirelles et al., 2001), while both forms of crossing can also result in the transmission of recipient oocyte mtDNA only (Meirelles et  al., 2001; Takeda et  al., 2003). Donor mtDNA has also been detected in porcine offspring (Takeda et  al., 2006) and in caprine embryos (Jiang et  al., 2004) derived by inter-specific SCNT (Takeda et al., 2006). Following cross-species (inter-generic) nuclear transfer, donor mtDNA can survive up to the 16-cell stage in human-bovine crosses (Takeda et  al., 2006), to the 20-cell stage in goat-sheep crosses (Bowles et  al., 2007), and persists in macaque-rabbit blastocysts (Yang et  al., 2003) and in human-rabbit SCNT-derived embryonic stem cells. Nevertheless, this outcome is indicative of the minority of embryos generated through inter-generic SCNT. Indeed, a fairer representation would be that, contrary to the predicted outcome, the donor cell preferentially interacts with the more divergent recipient oocyte mtDNA content, with donor cell mtDNA often being undetectable by the blastocyst stage (Bowles et al., 2007; Chang et al., 2003; Jiang et al., 2011).

WHY DOES THE DONOR CELL MITOCHONDRIAL DNA PERSIST? It is evident from embryos generated using intra- and interspecific ovine SCNT, where donor cells were depleted

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to 0.002% and 0.1% of their original mtDNA content or maintained their full mtDNA complement, that donor cell mtDNA can persist to the hatched blastocyst (Lloyd et al., 2006). This results from the continued expression of the donor cell mtDNA-specific replication factors, PolgA, PolgB, and TFAM, during pre-implantation development; this is unlike the expression patterns observed for these factors during pre-implantation development following in vitro fertilization (IVF) (Bowles et al., 2007). This continued expression occurs despite the depleted cells exhibiting increased population doubling times similar to those induced by serum starvation (Bowles et  al., 2007; Lloyd et  al., 2006). As a result, the donor cell has the potential to rescue its own population of mtDNA. As argued earlier, the introduction of any mitochondrial genetic material into an enucleated recipient oocyte has the potential to be transmitted to the offspring. For example, when embryonic cell nuclear transfer was performed with inter-specific crosses, mtDNA from the sperm contributing to the donor embryonic cell was also transmitted and inherited by the offspring (St John and Schatten, 2004). As donor cell mtDNA is propagated during preimplantation development and mtDNA copy number is not reduced in cloned embryos during this phase of development, donor cell mtDNA can be segregated at gastrulation, when there are no further reductions in copy number. As a result, it can then be selected for replication during organogenesis, when it can contribute to the overall mtDNA content of all or some of the tissues and organs of the developing offspring. While SCNT has been associated with a number of developmental abnormalities (Cibelli et  al., 2002) arising from aberrant epigenetic modifications (Morgan et  al., 2005), these factors also have implications for the premature expression of mtDNA during pre-implantation embryonic development. Recently, it has been demonstrated that embryonic stem cells derived through SCNT fail to establish the mtDNA set point (Kelly et al., 2011) as a result of their having very different patterns of Polg DNA methylation compared to embryonic stem cells derived from fertilized oocytes (Kelly et al., 2012).

TOXICITY OF SOMATIC MITOCHONDRIAL DNA From studies that have introduced somatic mitochondria, somatic cytoplasm, oocyte cytoplasm, or oocyte mitochondria into recipient oocytes, it is evident that somatic mitochondrial content has a deleterious effect on embryonic development (Takeda et  al., 2005). However, it has been demonstrated that there are improved developmental outcomes when the somatic cell is depleted of its mtDNA content during culture and then transferred into the recipient egg. In intra-specific crosses in sheep

SCNT, it is evident that depletion of the somatic mtDNA from the donor cell results in improved blastocyst rates (Lloyd et  al., 2006). In inter-generic SCNT, where, for example, a goat donor cell is depleted of its mtDNA content and then transferred into a sheep recipient oocyte, developmental outcomes are enhanced (Bowles et  al., 2007). Although there is no development to blastocyst, more of the embryos generated are able to develop further than the embryonic genome activation stage. These deleterious effects have two main causes. The first is the increased genetic diversity that results from two populations of mtDNA being mixed within a single embryonic entity (St John et  al., 2010). As there are strain and breed differences within a species for the mitochondrial genome, this results in some of the variants within coding genes encoding different amino acids (as demonstrated in inter-specific bovine (Steinborn et  al., 2002) and porcine (St John et  al., 2005) nuclear transfer offspring), which results in the formation of dysfunctional ETCs. This outcome is exemplified by cellular model systems in which enucleated somatic cells have been fused to mtDNAdepleted somatic cells. In more diverse rodent fusions, such as mouse-rat fusions, rat mtDNA can be replicated, transcribed, and translated by murine nuclear-encoded transcription and replication factors. However, ATP production is reduced (McKenzie et al., 2003; McKenzie and Trounce, 2000).

Somatic mitochondrial content has a deleterious effect on embryonic development.

Second, there is likely to be confusion between the host oocyte’s mtDNA population and the donor cell’s nucleus as to which population of mtDNA should be positively selected. While the mtDNA-specific POLG and the other key mtDNA replication factor, TFAM, are promiscuous, it is likely that they will not discriminate between populations of mtDNA to replicate but instead randomly select populations of mtDNA; hence, the heteroplasmic content within embryos and oocytes (Jiang et  al., 2011). This is probably the deciding factor in the random distribution of mtDNA content that is evident in cloned offspring. One approach does exist to overcome the transmission of donor cell mtDNA. It is feasible to deplete a donor cell of its mtDNA content during in vitro culture and then introduce this cell into the recipient oocyte. As with standard SCNT procedures, the reconstructed oocyte is activated and embryos are generated. The transfer of such embryos to surrogates produces offspring that inherit their mtDNA in exactly the same manner as do fertilized oocytes (Lee et al., 2010). This eliminates the nucleocytoplasmic conflicts that currently affect cloned offspring.

IS THERE A RELATIONSHIP BETWEEN DONOR CELL AND RECIPIENT OOCYTE MITOCHONDRIAL DNA THAT AFFECTS SOMATIC CELL NUCLEAR TRANSFER OUTCOME? It is evident from studies comparing the consequences of different mtDNA haplotypes on development that specific haplotypes can confer a specific advantage/disadvantage on development. For example, in a study comparing developmental outcomes for two breeds of cattle, one mtDNA haplotype demonstrated a greater efficiency in generating blastocysts following SCNT (Bruggerhoff et  al., 2002), while the other was more efficient in generating IVFderived embryos (Tamassia et  al., 2004). In a more comprehensive study analyzing the relationship between donor cell and recipient oocyte mtDNA following handmade cloning, it was evident that the donor cell favored the propagation of a slightly genetically more diverse mtDNA haplotype to that of its own (Bowles et al., 2008). This outcome was true to the blastocyst stage, during fetal development, and to term where the limits to mtDNA genetic divergence were approximately 0.07%. A similar outcome was observed for ovine SCNT, where approximately 0.04% divergence provided the outer limits for development to the blastocyst stage (Bowles et  al., 2007). Interestingly, success with inter-generic SCNT to the blastocyst stage requires the recipient oocyte’s mtDNA to be partially replaced with a more closely related population of mtDNA (Jiang et  al., 2011). Nevertheless, these experiments also identified key factors associated with pluripotency and chromosomal DNA replication that are species specific and essential to the reprogramming process. Consequently, there appears to be an optimum relationship between the mtDNA genetic divergence of the donor cell and recipient oocyte and development to term (see Figure 34.3).

CONCLUSION Although largely ignored as an important genome in the overall genetic context of cloning, the regulation of mtDNA transmission and replication is essential for successful development. For enhanced cloning outcomes, three criteria need to be addressed in the mtDNA context. First, donor cell mtDNA must be removed prior to the transfer to the nucleus to prevent dysfunctional ETCs from being generated. Second, the somatic cell must be effectively reprogrammed to prevent the premature replication of mtDNA during pre-implantation development that will impact on undifferentiated cells establishing the mtDNA set point. Third, the most appropriate mtDNA haplotype must be chosen to ensure that the appropriate chromosomal and mtDNA partners are matched. These are unlikely

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Birth Developmental outcome

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Oocyte mtDNA genetic distance between donor cell and recipient oocyte FIGURE 34.3  The importance of mitochondrial genetic distance to successful SCNT. The genetic distance between the donor cell and recipient oocyte is key to successful pre-implantation development to term. A small increase in genetic distance is optimal, while too great an increase, as with inter-generic SCNT results in poor developmental outcome. Modified from Kelly and St John (2010).

to be from the same source but rather from slightly more divergent sources. The requirement for slightly divergent recipient oocyte mtDNA has clear advantages for animal production; the chosen chromosomal genetic traits to be propagated can be done so with an mtDNA haplotype that offers other genetic traits, such as improved fertility, better meat quality, or ability to tolerate heat. This would allow, for example, beef cattle to be grazed in arid regions of the world, thus securing greater food supplies.

ACKNOWLEDGEMENT This work was supported by the Victorian Government’s Operational Infrastructure Support Program and Monash Institute of Medical Research start up funds to J.C.St.J.

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Chapter | 34  Mitochondrial DNA

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PART  | VI  SCNT as a Tool to Answer Biological Questions

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