Mitochondria in early development: linking the microenvironment, metabolism and the epigenome

in Reproduction
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  • 1 School of BioSciences, University of Melbourne, Parkville, Victoria, Australia

Correspondence should be addressed to A J Harvey; Email: ajharvey@unimelb.edu.au
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Mitochondria, originally of bacterial origin, are highly dynamic organelles that have evolved a symbiotic relationship within eukaryotic cells. Mitochondria undergo dynamic, stage-specific restructuring and redistribution during oocyte maturation and preimplantation embryo development, necessary to support key developmental events. Mitochondria also fulfil a wide range of functions beyond ATP synthesis, including the production of intracellular reactive oxygen species and calcium regulation, and are active participants in the regulation of signal transduction pathways. Communication between not only mitochondria and the nucleus, but also with other organelles, is emerging as a critical function which regulates preimplantation development. Significantly, perturbations and deficits in mitochondrial function manifest not only as reduced quality and/or poor oocyte and embryo development but contribute to post-implantation failure, long-term cell function and adult disease. A growing body of evidence indicates that altered availability of metabolic co-factors modulate the activity of epigenetic modifiers, such that oocyte and embryo mitochondrial activity and dynamics have the capacity to establish long-lasting alterations to the epigenetic landscape. It is proposed that preimplantation embryo development may represent a sensitive window during which epigenetic regulation by mitochondria is likely to have significant short- and long-term effects on embryo, and offspring, health. Hence, mitochondrial integrity, communication and metabolism are critical links between the environment, the epigenome and the regulation of embryo development.

Abstract

Mitochondria, originally of bacterial origin, are highly dynamic organelles that have evolved a symbiotic relationship within eukaryotic cells. Mitochondria undergo dynamic, stage-specific restructuring and redistribution during oocyte maturation and preimplantation embryo development, necessary to support key developmental events. Mitochondria also fulfil a wide range of functions beyond ATP synthesis, including the production of intracellular reactive oxygen species and calcium regulation, and are active participants in the regulation of signal transduction pathways. Communication between not only mitochondria and the nucleus, but also with other organelles, is emerging as a critical function which regulates preimplantation development. Significantly, perturbations and deficits in mitochondrial function manifest not only as reduced quality and/or poor oocyte and embryo development but contribute to post-implantation failure, long-term cell function and adult disease. A growing body of evidence indicates that altered availability of metabolic co-factors modulate the activity of epigenetic modifiers, such that oocyte and embryo mitochondrial activity and dynamics have the capacity to establish long-lasting alterations to the epigenetic landscape. It is proposed that preimplantation embryo development may represent a sensitive window during which epigenetic regulation by mitochondria is likely to have significant short- and long-term effects on embryo, and offspring, health. Hence, mitochondrial integrity, communication and metabolism are critical links between the environment, the epigenome and the regulation of embryo development.

Introduction

Mitochondria are double membrane-bound organelles that generate ATP through oxidative phosphorylation (OXPHOS), the components of which are partially encoded by their own genome. They represent the most abundant organelle in the oocyte and undergo significant structural and positional changes during preimplantation development (Motta et al. 2000, Sathananthan & Trounson 2000). This dynamic nature is essential for modulating key cellular events, coincident with the changing energy requirements of the embryo (Gardner 1998), yet the significance of mitochondria during preimplantation embryo development extends beyond the generation of ATP. This review integrates recent advances in our understanding of the dynamics of mitochondria, and the interactions between mitochondria and the cytoskeleton, and other organelles, that ensure mitochondrial, and oocyte and embryo, integrity and facilitate signalling. The implications of deficits in mitochondrial dynamics and signalling for embryo viability and offspring health are also explored, with an emphasis on the emerging understanding that modulation of metabolic flux contributes to the regulation of the nuclear epigenetic landscape (Donohoe & Bultman 2012, Harvey et al. 2016), proposing a novel role for mitochondria as mediators of metaboloepigenetic processes that programme the oocyte and preimplantation embryo. Consequently, the utility of mitochondria as a biomarker is limited by deficiencies in our understanding of the physiological and cellular consequences of in vivo and in vitro manipulation of mitochondrial function, and the link between mitochondrial movement and metabolite signalling during preimplantation embryo development that mediate alterations in the epigenome.

Mitochondrial function and cellular homeostasis

Mitochondria are primarily recognised as the powerhouse of cells, synthesising ATP through OXPHOS. The electron transport chain (ETC) comprises five complexes (I–V) that regulate the transfer of nicotinamide adenine dinucleotide (NADH) and succinate through the ETC, and the generation of an electrochemical gradient across the inner mitochondrial membrane to drive ATP synthase (complex V). Respiratory activity is determined by the assembly of respiratory chain complexes into functional supercomplexes within the inner mitochondrial membrane (Cogliati et al. 2013), although single complexes are also believed to exist to enable flexibility and adaptation to environmental changes (Porras & Bai 2015). The environment created within the inner mitochondrial membrane is distinct from the cytosol (Tzagoloff 1982, Herrmann & Riemer 2010), hence the mitochondrion possesses shuttles which regulate specific metabolite levels between the cytoplasm and the mitochondria and contribute to metabolic homeostasis (Herrmann & Riemer 2010, Wang et al. 2014). Mitochondrial metabolism also plays a role in the synthesis of macromolecules, such as fatty acids, amino acids and nucleotides, to support proliferation (reviewed by Ahn & Metallo 2015). These diverse functions underscore the significance of mitochondria as regulators of cellular metabolism, however mitochondrial function extends beyond the traditional confines of metabolic regulation.

Historically, mitochondria have been perceived as signalling effectors, that is, mere bystanders in the response to various stimuli. However, mitochondria are increasingly viewed as active participants in numerous pathways, acting as initiators and transducers of signals through the modulation of metabolite availability and changes in redox state (Chandel 2014). Beyond the production of ATP via OXPHOS, mitochondria participate in numerous cellular functions, including Ca2+ homeostasis (Duchen 2000, Rizzuto et al. 2012), stress responses (Zhao et al. 2002, Jovaisaite et al. 2014), apoptosis (reviewed by Tait & Green 2013) and have been proposed to be involved in the regulation of chromosome segregation (Schon et al. 2000, Qian et al. 2012). A consequence of OXPHOS is the production of reactive oxygen species (ROS), yet in stark contrast to the damaging effects of ROS at high concentrations, physiological levels are necessary to act as signalling molecules to directly modulate numerous processes through the modification of kinases, transcription factor activity, growth factors and metabolic enzymes (reviewed Harvey et al. 2002, by Hamanaka & Chandel 2010, Lees et al. 2017). Consequently, mitochondria are central to cellular and metabolic homeostasis and are key players in diverse physiological (and pathological) processes.

Importantly, mitochondrial function has also been connected to cell specification (Chung et al. 2007), where lineage commitment necessitates functional mitochondrial adaptation to support differentiation. Cellular energy demand varies significantly depending on cell function and activity, therefore modulation of energy production to accommodate changing physiological demands is essential and likely underpins cell identity. Indeed, once considered mere by-products of cellular metabolism, recent evidence supports a role for mitochondrial metabolites as signalling molecules that regulate gene expression, through the regulation of epigenetics, and consequently drive lineage decisions (Shyh-Chang et al. 2013a, Folmes & Terzic 2014, Harvey et al. 2016). Mitochondrial activity is an absolute requirement for stem cell differentiation, as inhibiting mitochondrial activity with antimycin A or CCCP (carbonyl cyanide m-chlorophenyl hydrazine) prevents embryonic stem cell (ESC) differentiation (Mandal et al. 2011, Pereira et al. 2013). Mitochondrial metabolism in particular controls the levels of the key co-factors including acetyl-CoA, α-ketoglutarate (αKG) and NADH/NAD+, as well as other tricarboxylic acid (TCA) intermediates including citrate and succinate, which act as key substrates for epigenetic modifiers (Wallace & Fan 2010). Studies in ESC have begun to highlight the significance of nutrient regulation in establishing the epigenome (reviewed by Harvey et al. 2016), however, our understanding of the consequences of modulating nutrient and metabolic intermediate levels on the epigenetic landscape in the oocyte, and during preimplantation embryo development, remains rudimentary.

Dynamic regulation of the preimplantation epigenome

Preimplantation embryo development is characterised by extensive reprogramming of the epigenetic landscape to support and regulate stage-specific events including embryonic genome activation and lineage specification. Following fertilisation, the paternal genome undergoes a wave of active DNA demethylation (Mayer et al. 2000, Oswald et al. 2000). Subsequently, passive demethylation proceeds with successive cleavage divisions, persisting through to the morula stage (Monk et al. 1987, Santos et al. 2002, Zaitseva et al. 2007), with the exception of imprinted genes and some repetitive elements. Formation of the blastocyst results in the establishment of the first cell lineages, in which the pluripotent inner cell mass (ICM) exhibits higher levels of de novo methylation compared with the trophectoderm (Reik et al. 2003). In addition to DNA methylation, chromatin remodelling and histone modifications also play a critical role in programming the embryonic epigenetic landscape. However, in contrast to DNA methylation, the dynamics of acetylation (ac) and histone methylation (me) during early development is largely limited to studies on the expression of histone (de)acetylases, methyltransferases and demethylases (reviewed Shi & Wu 2009, by Mason et al. 2012). Immunofluorescent analyses have however documented asymmetry in a number of pronuclear modifications (Adenot et al. 1997, Santos et al. 2005, van der Heijden et al. 2005, Boskovic et al. 2012), along with the presence of other marks in both pronuclei (Zhou et al. 2014, Rollo et al. 2017) and stage-specific changes (Ma & Schultz 2008, Boskovic et al. 2012, Liu et al. 2016b). Consequently, normal development relies on coordinated changes in DNA methylation, and histone acetylation and methylation, which underpin the activation and silencing of specific genes (reviewed by Santos & Dean 2004, Lucas 2013, Messerschmidt et al. 2014, Marcho et al. 2015).

Aberrant epigenetic patterns have been correlated with alterations in the microenvironment surrounding the developing embryo, as well as with subsequent developmental failure (Lucas 2013, Vickaryous & Whitelaw 2005, Ma & Schultz 2008)). In vitro embryo culture has been shown to accelerate DNA demethylation of rabbit embryos (Reis e Silva et al. 2012) and significantly lower histone 3 lysine 4 trimethylation (H3K4me3; Wu et al. 2012), while exposure of in vivo derived bovine embryos to in vitro culture conditions induces significant hypermethylation or hypomethylation depending on the timing of exposure, and include alterations in metabolic pathways (Salilew-Wondim et al. 2018). Further, increased acetylation of histone 3 lysine 9 (H3K9ac) has recently been proposed as a biomarker of culture-induced perturbations (Rollo et al. 2017). Importantly, inhibition of histone deacetylase activity with trichostatin A reduces (Adenot et al. 1997) or inhibits (Ito et al. 2000) blastocyst development, and perturbations in the programming of the preimplantation epigenome have been shown to reduce implantation (Ding et al. 2012) and disturb post-implantation decidualisation (Gao et al. 2012). While it remains to be determined whether epigenetic perturbations persist into foetal tissues, combined, these studies reveal that the preimplantation embryonic epigenome is sensitive to alterations in the surrounding nutrient environment, with alterations negatively impacting on subsequent development. Significantly, the activity of epigenetic modifiers that regulate the deposition and removal of DNA and histone marks depends on the availability of specific metabolites. Conceivably, the changing energy requirements, and therefore metabolic pathway activity, of the preimplantation embryo may drive epigenetic programming, such that perturbed metabolic activity transmits alterations in the cellular microenvironment which modify the epigenetic landscape (Gardner & Harvey 2015).

Metabolic regulators of acetylation and methylation

Levels of histone acetylation are modulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation by histone acetyltransferases requires acetyl-CoA, a metabolic intermediate whose availability dictates histone acetylation levels, and consequently gene transcription. High glycolytic rates drive acetyl-CoA synthesis in mitochondria, although acetyl-CoA can also be produced in the cytosol from citrate, fatty acid and ketone body oxidation and the catabolism of amino acids, or within the nucleus (Wellen et al. 2009). Recently, glucose flux through glycolysis has been shown to regulate ESC acetyl-CoA levels, and therefore histone acetylation (Moussaieff et al. 2015). Supplementing human ESC with acetate, a precursor of acetyl-CoA, increases acetylation levels, blocks histone deacetylation and reduces differentiation, while inhibition of acetyl-CoA production from glucose results in the loss of pluripotency (Moussaieff et al. 2015). Alterations in glucose use can therefore significantly modify not only downstream metabolite levels, but also alter histone acetylation, through altered acetyl-CoA levels, and subsequently regulate differentiation. The significance of this is such that incorrect timing of substrate availability in the early embryo may accelerate or delay key events through gene activation and is particularly relevant given the changing energy requirements of the preimplantation embryo (reviewed by Gardner & Harvey 2015) and the significance of glucose use as a marker of human embryo viability (Gardner et al. 2011). As alterations in glucose uptake can significantly alter acetyl-CoA availability, it will be important to understand the impact of altering acetyl-CoA levels during preimplantation development. A role for appropriate modulation of acetyl-CoA levels to support embryo development, potentially via the epigenome, is however plausible given the identification of the embryotrophic factor promoting blastocyst development within bovine serum albumin as the acetyl-CoA precursor citrate (Gray et al. 1992).

In contrast, repression of gene transcription is in part mediated through histone deacetylation. In addition to Class I and II HDACs, which are dependent on zinc as a co-factor for their activity, nicotinamide adenine dinucleotide (NAD+) availability regulates a family of conserved NAD+-dependent histone deacetylases known as sirtuins (SIRTs), which deacetylate targets involved in the modulation of cellular metabolism and mitochondrial biogenesis, cell survival, DNA repair, differentiation and ROS production (Houtkooper et al. 2012). Of note, alterations in sirtuin activity have been shown to alter embryo development. Inhibition of sirtuin activity with nicotinamide or sirtinol significantly reduces porcine (Kwak et al. 2012) and mouse (Kawamura et al. 2010) blastocyst development, conceivably through alterations in acetylation. It will however be important to examine the regulation of sirtuins, and sirtuin regulated changes in the epigeneome, during preimplantation development in response to nutrient availability, particularly given the expanded use of monophasic culture systems in preference to biphasic media. Equally, investigation of the significance of NAD+ in regulating sirtuin activity during preimplantation development is warranted. NAD+ is synthesised de novo from the amino acid tryptophan (Bender 1983) or recycled via the NAD+ salvage pathway from nicotinamide and derivatives of vitamin B3 (Canto et al. 2015). In order to ensure the continuation of glycolysis, NAD+ is also recycled via the conversion of pyruvate to lactate, the production of which increases with blastocyst development (Gardner & Harvey 2015). NADH reducing equivalents are also shuttled across the impermeable inner mitochondrial membrane via the malate aspartate shuttle (MAS) to maintain NAD+:NADH (Lu et al. 2008). Significantly, the activity of the MAS is responsible for oxidative metabolism during embryo development (Lane & Gardner 2005) and inhibition of MAS activity with amino-oxyacetate impairs embryo metabolism and ATP production, accompanied by reduced blastocyst development and placental and foetal growth (Mitchell et al. 2009, Wakefield et al. 2011). Whether these changes are accompanied by alterations in the foetal epigenome remains to be determined. These studies however highlight a role for NAD+ regulation in programming development, and a requirement for coordinated compartmentalisation of metabolites between the cytoplasm and mitochondria to maintain mitochondrial function and ensure normal development.

In a similar manner, the activity of DNA methyltransferases (DNMTs), which catalyse the addition of methyl groups to the 5′ position of cytosine residues within cytosine-guanine dinucleotides (CpG) and result in the formation of 5-methylcytosine (5mC), as well as histone methyltransferases which catalyse the transfer of 1 (me), 2 (me2) or 3 (me3) methyl groups to lysine and arginine residues on histones, rely on metabolic intermediate availability. The amino acid methionine is the precursor of the universal methyl donor S-adenosylmethionine (SAM), catalysed by methionine adenosyltransferases (MATs), that acts as a co-factor in methylation reactions (Finkelstein 1990). Methionine has been found to be essential for mouse and bovine blastocyst formation, as omission of methionine from culture reduces mouse blastocyst formation, and morula and blastocyst H3K4me3 (Sun et al. 2018), while addition of the methionine antagonist ethionine following embryonic genome activation (EGA) impairs blastocyst development and cell number (Menezo et al. 1989, Ikeda et al. 2012, Kudo et al. 2015) accompanied by a reduction in H3K9me3 modification of Nanog and Tead4 (Kudo et al. 2015). Importantly, this loss in developmental potential coincides with the increase in SAM synthesis that occurs following compaction (Menezo et al. 1989), suggesting a requirement for appropriate metabolite availability at the time of EGA. A requirement for SAM is also evidenced by studies that demonstrate that inhibition of MAT2A, which catalyses the formation of SAM, significantly reduces bovine and mouse blastocyst development and cell number and increases apoptosis (Ikeda et al. 2017, Sun et al. 2018). Conversely, SAM supplementation during bovine embryo culture enhances hatching, accompanied by significant alterations in the global DNA methylation landscape (Shojaei Saadi et al. 2016). Likewise, methionine deprivation reduces SAM levels in human ESC, accompanied by a rapid decrease in H3K4me3 and reduction in global DNA methylation, which can be abrogated through SAM supplementation, with changes in methylation potentiating subsequent differentiation (Shiraki et al. 2014). These data reveal that methionine is important not only for maintaining the pluripotent epigenome but its availability has the potential to alter the timing of lineage specification.

Maternal methionine supplementation has likewise been associated with alterations in bovine blastocyst gene expression (Penagaricano et al. 2013), demonstrating that even subtle changes in methionine availability can impact development. Indeed, methionine utilisation has been proposed as a biomarker of human embryo developmental potential (Houghton et al. 2002). Considerably, deficiencies in metabolites that intersect the folate and methionine cycles, including folate (O’Neill 1998, Kwong et al. 2010) and vitamin B12 (Sinclair et al. 2007), are also known to impact embryo development, and more significantly, alter post-natal growth and health and DNA methylation (Sinclair et al. 2007). Threonine, metabolised via the folate cycle, likewise regulates SAM levels in mouse ESC (Wang et al. 2009a, Shyh-Chang et al. 2013b). The cytoplasmic folate cycle intersects with mitochondrial formate availability (Bao et al. 2016), while mitochondrial NADH oxidation, and therefore TCA activity, has recently been linked to methionine metabolism (Lozoya et al. 2018), highlighting a link between mitochondrial activity and methylation. Mitochondrial one-carbon metabolism therefore integrates nutrient, amino acid metabolism and mitochondrial activity, such that differences in pathway flux can profoundly alter the epigenome.

Conversely, α-KG, a product of the oxidation of glucose and glutamine via the mitochondrial TCA not only modulates the activity of Ten-Eleven translocation (TET) dioxygenases (Xiao et al. 2012) but also regulates the activity of Jumanji (JMJ) demethylases which remove methyl groups from lysine residues (Tsukada et al. 2006). The TET family of enzymes (TET1, 2 and 3) use oxygen to catalyse the oxidative decarboxylation of α-KG, generating a reactive enzyme-bound intermediate which converts 5mC to 5-hydroxymethylcytosine (5hmC). At the pronuclear stage, Tet3 mediates active demethylation of the paternal genome (Gu et al. 2011, Iqbal et al. 2011, Wossidlo et al. 2011) with 5hmC progressively lost through to the 8-cell stage (Inoue & Zhang 2011). In contrast, H3K9me2 of maternal chromatin enables the recruitment of Dppa3 (also known as Stella) to exclude Tet3 from binding and enabling its passive demethylation (Wossidlo et al. 2011, Nakamura et al. 2012). Tet3 expression in porcine embryos is regulated by EGA and modulates Nanog (Gu et al. 2011, Lee et al. 2014b), suggesting an involvement in lineage specification. Tet1 is detectable throughout preimplantation development in the mouse (Ito et al. 2010) and from the 4-cell stage in porcine embryos (Lee et al. 2014b) and becomes enriched in the ICM compared with the trophectoderm at the blastocyst stage, being absolutely required for specification toward the ICM compartment (Ito et al. 2010). The differential expression pattern plausibly reflects the differences in metabolism between these two lineages. Indeed, glucose- and glutamine-derived α-KG modulate mouse ESC histone and TET-dependent DNA demethylation (Carey et al. 2015), suggesting that glycolytic activity supports DNA demethylation and plausibly that appropriate uptake of glucose is likely necessary to establish correct (de)methylation dynamics. This may in part explain the significance of the relationship between glucose uptake and embryo viability (Gardner & Leese 1987, Gardner et al. 2011). Indeed, in vitro culture has been associated with increased DNA methylation relative to in vivo derived embryos in both the mouse and rat (Zaitseva et al. 2007), suggesting dysregulation of demethylating enzymes. It will therefore be important to determine whether modulation of α-KG impacts embryo development, accompanied by changes in methylation.

Appropriate regulation of histone demethylation is also required for normal preimplantation embryo development, mediated by JMJ demethylases which remove the methyl groups from lysine residues in histones in a Fe(2+) and α-KG-dependent manner. Knockdown of maternal JMJD3 (also known as KDM6B), required for the demethylation of H3K27me3 from the 2-cell stage, significantly reduces mouse (Canovas et al. 2012) and bovine blastocyst development accompanied by a reduction in both ICM and trophectoderm cell numbers (Chung et al. 2017), with JMJD3 activity correlated with α-KG availability in mouse ESC (Carey et al. 2015). Moreover, knockdown of JMJD2C (Wang et al. 2010) and the JmjC demethylase KDM5B (Huang et al. 2015) impairs mouse and porcine blastocyst development respectively, accompanied by perturbations in histone methylation, while inhibition of H3K4 demethylation impairs mouse embryo cleavage at the time of EGA (Shao et al. 2008), further indicating that appropriate, stage-specific regulation of the epigenetic landscape is required for development.

Combined, these studies identify that metabolism, and in particular mitochondrial activity, acts as a primary interface between the environment and the epigenome (Fig. 1). As a consequence, perturbations in metabolic pathway activity alter the production and availability of co-factors required for epigenetic modifier activity, resulting in an altered, and heritable, nuclear epigenetic landscape (reviewed by Donohoe & Bultman 2012, Brown et al. 2015, Harvey et al. 2016) at the time of lineage specification when the epigenetic landscape is being programmed. Therefore, perturbations in the nutrient environment surrounding the oocyte and preimplantation embryo may modulate long-term offspring health and viability through alterations in metabolism that impact the programming epigenome. However, it is also important to consider the dynamic nature of mitochondria within the oocyte and early embryo, as a means to deliver not only ATP but also facilitate and regulate the deposition and removal of epigenetic modifications in a stage-specific manner.

Figure 1
Figure 1

Mitochondrial regulation of the preimplantation epigenome. Diet and the extracellular microenvironment regulate the availability of nutrients that modulate metabolic pathway activity and mitochondrial metabolism, regulating the supply of intermediates that act as co-factors for epigenetic modifying enzymes. Glycolytic flux and the activity of the mitochondrial malate aspartate shuttle (MAS) determines the NAD+:NADH ratio. NAD+ regulates the activity of the NAD+-dependent histone deacetylases (sirtuins), repressing gene transcription. Conversely, acetyl-CoA, generated from glucose-derived pyruvate or β-oxidation of fatty acids, acts as a co-factor for histone acetyltransferases (HAT) responsible for transcriptional activation. Similarly, alpha-ketoglutarate (αKG) derived from the mitochondrial tricarboxylic acid (TCA) cycle and glutamine (Gln) metabolism mediates histone and DNA demethylation catalysed by Jumanji (JMJ) and Ten-Eleven Translocation (TET) demethylases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) respectively, activating transcription. Folate-mediated one-carbon metabolism relies on a dietary source of folate to supply tetrahydrofolate (THF), as well as serine (Ser) or mitochondrial-derived formate for the supply of 1C units. Methionine (Met) metabolism is required for S-adenosylmethioninine (SAM) generation via the folate and methionine cycles. SAM acts as a methyl donor for histone (HMT) and DNA (DNMT) methyltransferases to mediate transcriptional repression. Beyond their historical role in providing ATP (adenosine triphosphate), the dynamic nature of mitochondria during the development of the oocyte and preimplantation embryo likely establishes nutrient gradients that finely programme the preimplantation epigenetic landscape to mediate key developmental events, including lineage specification. Consequently, perturbations in nutrient availability, mitochondrial activity or mitochondrial movement have the potential to significantly alter long-term development through the regulation of heritable changes in the epigenome prior to differentiation. 5-mTHF, 5-methyl tetrahydrofolate; ac, acetylation; Asp, aspartate; Glu, glutamate; Gly, glycine; GSH, glutathione; HCY, homocysteine; me, methylation; NAD+, nicotinamide adenine dinucleotide; NADH, reduced form of NAD+; OAA, oxaloacetate; OXPHOS, oxidative phosphorylation; SAH, S-adenosylhomocysteine; THF, tetrahydrofolate.

Citation: Reproduction 157, 5; 10.1530/REP-18-0431

Mitochondrial dysfunction and fertility

In order to support the dynamic changes that occur during preimplantation embryo development, mitochondria within the oocyte and early embryo have primarily been regarded as necessary to generate adequate ATP to fuel the first few days of embryo development (Dumollard et al. 2004). In line with this, the mammalian preimplantation embryo undergoes significant changes in its metabolism during development, where metabolic pathway activity and ATP content vary significantly between human oocytes and embryos (Leese et al. 1986, Chi et al. 1988, Hardy et al. 1989, Gott et al. 1990, Slotte et al. 1990, Conaghan et al. 1993, Gardner & Harvey 2015). Growing oocytes preferentially metabolise pyruvate via OXPHOS, while their somatic compartment, the cumulus cells, are more glycolytic, metabolising glucose to supply the pyruvate required by the oocyte (reviewed by Dumesic et al. 2015). Similarly, the early embryo is characterised by low levels of oxidative metabolism and oxygen consumption, utilising pyruvate, lactate and amino acids to support development (Houghton et al. 1996, Gardner & Wale 2013). In contrast, with the activation of the embryonic genome and the increased energy demand required for blastocoel formation, the blastocyst stage embryo exhibits both high levels of glycolysis and oxygen consumption (reviewed Mills & Brinster 1967, Wales 1986, Houghton et al. 1996, Thompson et al. 1996, by Gardner & Harvey 2015). These changing energy requirements therefore necessitate dynamic and coordinated regulation of metabolism to support ongoing development.

In further support of a requirement for mitochondrial ATP production during development, perturbations in mitochondrial activity have been found to impact embryo development and implantation potential (Van Blerkom et al. 1995), as well as foetal and placental growth (Wakefield et al. 2011). Oocytes containing higher levels of ATP result in significantly higher fertilisation rates and increased blastocyst development. Conversely, those deficient in ATP result in compromised development (Stojkovic et al. 2001, Nagano et al. 2006), reflecting the need for sufficient ATP to support embryo development. A reduced capacity of oocytes to generate ATP has been observed in aged mouse and hamster oocytes (Simsek-Duran et al. 2013) and has been associated with abnormal nuclear spindle and chaotic chromosomal distribution in humans (Wilding et al. 2003) with oocyte mitochondrial activity in particular known to decrease linearly with increasing age (Wilding et al. 2001), and vary with stimulation regimen (Cree et al. 2015). Alterations in ATP content manifest not only as poor or abnormal human oocyte quality but also as reduced embryo development and pregnancy rates (Van Blerkom et al. 1995). However, this represents an oversimplistic view of the impact of mitochondrial activity on the maturing oocyte and developing embryo and fails to consider the importance of these organelles in modulating other aspects of cell homeostasis.

An alternative explanation for reduced fertility with ageing, or environmental perturbations, beyond ATP availability, is the accompanying alteration in the provision of metabolic intermediates necessary for modulation of the epigenetic landscape. Indeed, oocyte ageing is accompanied by reduced 5mC and DNMT expression levels (Yue et al. 2012), H4K12 and H4K16 acetylation (Manosalva & Gonzalez 2009) and histone methylation (Manosalva & Gonzalez 2010). Taken together, it is plausible that alterations in metabolic activity mediate the changes in the epigenetic landscape of oocytes and/or the early embryo, impacting subsequent developmental competence. Surprisingly, relatively few studies have investigated specific reproductive tract metabolite levels associated with ageing or dietary changes. Alterations in human follicular fluid lipid profiles have been associated with ageing (Cordeiro et al. 2018), as well as with embryos that fail to cleave (O’Gorman et al. 2013), however whether epigenetic change is elicited by these environments remains unclear. Ageing likewise alters cumulus cell function, with a significant impact on transcripts (Al-Edani et al. 2014) and proteins (McReynolds et al. 2012) involved in metabolism, possibly reflecting a reduced ability to support the metabolic and epigenetic requirements of the oocyte. As acetylation has recently been shown to be regulated by lipids and lipid-derived acetyl-CoA (McDonnell et al. 2016), and circulating maternal lipids have been shown to programme the developing foetal DNA methylation landscape (Marchlewicz et al. 2016), alterations in follicular fluid lipid content with ageing may perturb the oocyte epigenetic landscape. Indeed, lipid-derived acetyl-CoA is more readily incorporated into histones than that derived from glucose (McDonnell et al. 2016), emphasising that metabolic plasticity results in epigenetic remodelling in response to nutrient availability. Oocyte NADH/FAD levels have also been correlated with ageing (Babayev et al. 2016), and mitochondrial dysfunction has been associated with reduced NADH/NADPH in human zygotes (Wilding et al. 2001, 2002, 2003), yet whether such changes impact oocytes from young individuals, or the regulation of the sirtuin NAD+-dependent deacetylases, remains to be determined. The effects of ageing on the levels of other metabolites within follicular or reproductive tract fluid, including acetyl-CoA, have similarly not been investigated. It will therefore be essential to examine metabolite perturbations associated with poor developmental competence and outcomes, along with consequences for long-term offspring health, to establish whether specific changes in metabolite content associated with ageing and nutrient availability result in epigenetic priming that plays a role in the reduction in developmental competence.

Mitochondrial morphology in the oocyte and preimplantation embryo

During preimplantation embryo development, mitochondria undergo dynamic, functional and structural changes, morphing from spherical organelles containing dense matrices and few peripheral arched cristae to long filamentous organelles with numerous transverse cristae that maximise the surface area available for OXPHOS (Zick et al. 2009). This contrasts with the reticular, elongated, electron- and cristae-rich morphology of somatic cells. A spherical morphology is also characteristic of ESC (St John et al. 2005, Cho et al. 2006, Chung et al. 2007), suggesting that it underpins the pluripotency of these cells (reviewed by Harvey et al. 2013). These mitochondrial changes therefore reflect the developmental stage, and metabolic requirements, of the embryo (reviewed by Bavister & Squirrell 2000, Lees et al. 2017), but more significantly likely also facilitate mitochondrial signalling and crosstalk.

The primary human oocyte contains many spherical mitochondria with few peripheral arched cristae dispersed throughout the cytoplasm (Motta et al. 2000). Mitochondria remain primarily spherical during folliculogenesis, with pale matrices and small vesicular cristae, clustered around the nucleus (Stern et al. 1971, Sathananthan & Trounson 2000, Van Blerkom 2004). While primordial oocytes contain approximately 10,000 mitochondria, this increases to around 100,000 in mature oocytes (Lanzavecchia & Mangioni 1964, Sathananthan et al. 1986, Jansen & de Boer 1998). By ovulation, mitochondria are the most prominent organelle in the oocyte cytoplasm (Motta et al. 2000). During early cleavage, elongated mitochondria with well-developed transverse cristae begin to emerge (Piko & Matsumoto 1976, Motta et al. 2000). As development proceeds, the proportion of elongated, cristate-rich mitochondria increases, such that by the blastocyst stage in the human and mouse, blastomeres contain a mixed population of predominantly spherical and elongated mitochondria in the ICM and trophectoderm respectively (Nadijcka & Hillman 1974, Mohr & Trounson 1982, Motta et al. 2000, Sathananthan & Trounson 2000, Lees et al. 2017).

Despite the perceived ‘immature’ morphology, oocyte mitochondria are highly active, producing the majority of energy required to support development. The structural changes are however essential for preimplantation embryo development to proceed (Van Blerkom 1989) and are consistent with a progressive activation of mitochondrial respiratory activity, inferred from increased oxygen consumption and ATP production via OXPHOS (Houghton et al. 1996, Thompson et al. 1996, Trimarchi et al. 2000, Sturmey & Leese 2003) and changes in cristae shape (Mohr & Trounson 1982, Sathananthan & Trounson 2000, Cogliati et al. 2013). Interestingly, human apical trophectoderm cells are characterised by elongated mitochondria with transverse cristae, largely located around the periphery (Mohr & Trounson 1982). This morphological difference in both mitochondrial architecture and activity likely reflects differences in energy demand, but may also facilitate the process of implantation or may play a role in regulating cell specification, plausibly by differential metabolite availability regulating the epigenome. Metabolites are increasingly recognised as regulators of pluripotency and cell fate (Chung et al. 2007, Shyh-Chang et al. 2013b, Shiraki et al. 2014, Moussaieff et al. 2015), with mitochondria at the heart of sensing and regulating lineage decisions and establishing cellular memory (reviewed by Harvey et al. 2016, Lees et al. 2017). It is tempting to speculate that mitochondria may be involved in the regulation of the earliest embryonic specification event, the establishment of a polarised epithelium, in a manner similar to that observed when hepatocytes initiate polarisation, necessitating mitochondrial elongation (Fu et al. 2013). Notably, ATP generated via OXPHOS is required for this process (Fu et al. 2013). Further, the Hippo pathway, which mediates the differentiation of the trophectoderm from the ICM (reviewed by Manzanares & Rodriguez 2013), also regulates mitochondria fusion and size (Nagaraj et al. 2012). These data suggest that mitochondrial structure and activity, and cell polarisation, may be intricately linked, such that deficiencies in mitochondrial dynamics and activity during early development may have a significant impact on cell allocation and developmental capacity beyond the provision of ATP.

Regulation of mitochondrial morphology

Mitochondrial morphology adapts to fluctuating cellular requirements by modulating changes in shape through fission and fusion (Frederick & Shaw 2007), continually dividing and fusing to form a dynamic interconnecting network necessary to maintain mitochondrial and mtDNA integrity (Suen et al. 2008). Fission and fusion events are mediated by a number of nuclear encoded proteins (reviewed by Wai & Langer 2016). Mitochondrial fusion not only enables the creation of extensive mitochondrial networks to increase energy supply (Chen et al. 2005) but also the complementation, and the mixing of mitochondrial content, between healthy and defective mitochondria (Youle & van der Bliek 2012). The outer mitochondrial membrane GTPase required for mitochondrial fusion; fuzzy onions (fzo; Hales & Fuller 1997) was identified as an essential regulator of spermatogenesis. The human homologs, mitofusin 1 and 2 (MFN1, MFN2 respectively) likewise display GTPase-dependent activity and mediate mitochondrial fusion (Santel & Fuller 2001, Chen & Chan 2004). Cells deficient in either Mfn1 or Mfn2 display considerably reduced levels of mitochondrial fusion and distinct types of fragmented mitochondria (Chen et al. 2003, 2005) and increased ROS (Lee et al. 2014a). Beyond their role in spermatogenesis, they also contribute to ongoing development. Mice deficient in either Mfn1 or Mfn2 die around embryonic day (E) 11.5. Mfn1 mutants are significantly smaller with a pronounced developmental delay prior to resorption, while Mfn2 mutant embryos display a severe disruption of the placental trophoblast giant cell layer (Chen et al. 2003). Mfn2 knockdown in oocytes reduces oocyte maturation and fertilisation and alters mitochondrial distribution and spindle morphology (Liu et al. 2016a), further implicating mitochondria in the regulation of chromosome segregation in the oocyte. Moreover, reduced Mfn2 expression following siRNA attenuates mouse blastocyst formation and significantly reduces the number of embryos progressing through the third cell division (Zhao et al. 2015). As this coincides with the activation of the embryonic genome, active mitochondrial fusion is likely required to support this milestone. Embryos that survive Mfn2 knockdown, exhibit decreased ATP and mtDNA levels, reduced mitochondrial membrane potential and increased levels of apoptosis (Zhao et al. 2015), further suggesting that adequate control of mitochondrial dynamics is necessary for continued development, however whether these knockout phenotypes were accompanied by alterations in the epigenome, particularly at the time of EGA, were not examined.

A third protein, Optic Atrophy 1 (OPA1), is also essential for inner mitochondrial membrane fusion (Olichon et al. 2003). Opa1 knockout is similarly embryonically lethal by E13.5 in the mouse, further supporting a role for mitochondrial dynamics in regulating development (Davies et al. 2007). Loss of OPA1 results in cristae widening, thereby playing a role in the regulation of mitochondrial cristae shape (Patten et al. 2014). Significantly, low ADP concentrations, characteristic of the late cleavage and morula stage embryo (Leese et al. 1984), were reported to modify cristae shape to a more expanded, less dense matrix more than 50 years ago (Hackenbrock 1966), however only recently has cristae shape and volume been found to modulate the rates of several TCA cycle enzymes (Lizana et al. 2008), as well as the mitochondrial membrane, redox-sensitive protein ROS modulator 1 (ROMO1; Norton et al. 2014) and cardiolipin (Ikon & Ryan 2017, reviewed by Quintana-Cabrera et al. 2018). How these pathways are regulated by changes in nutrient availability, particularly during embryo development, remains to be determined. Given the redox-sensitive nature of OPA1 (Garcia et al. 2018) and ROMO1, ROS, NAD+:NADH and ATP:ADP ratios, which are impacted by in vitro and in vivo nutrient availability, are likely important signalling molecules that link mitochondrial metabolism, cristae morphology and cell signalling. Conceivably, any form of oxidative stress or metabolic dysfunction induced by assisted reproductive technologies, including culture, may therefore interfere with mitochondrial dynamics.

Conversely, the process of mitochondrial fission is mediated in part by dynamin-related protein 1 (Drp1), a large GTPase and fission 1 (Fis1). Drp1 is a large dynamin-related GTPase located primarily in the cytoplasm, with punctuate localisation marking future sites of fission (Smirnova et al. 2001). Downregulation of Drp1 results in loss of mitochondrial DNA, decreased mitochondrial respiration and increased ROS production in HeLa cells (Parone et al. 2003), as well as reduced cell proliferation and cellular ATP. Knockout of Drp1 in mice leads to embryonic lethality at E11.5 accompanied by a reduction in size, plausibly attributable to the loss of placental giant cells (Ishihara et al. 2009, Wakabayashi et al. 2009). Perturbations in placental structure would contribute to reduced nutrient transport, thereby implicating mitochondria in regulating placental cell differentiation and fate. In addition, oocyte specific Drp1 knockout results in infertility (Udagawa et al. 2014), and both decreased expression of Drp1 and increased Mfn2 mRNA levels, favouring mitochondrial fusion, have been associated with fragmented human day 3 embryos (Otasevic et al. 2016), although whether this is a cause or consequence is unclear. Further, inhibition of mitochondrial fission with the specific Drp1 inhibitor Mdivi-1 reduces porcine blastocyst formation accompanied by reduced mitochondrial membrane potential and increased ROS (Yeon et al. 2015), with increasing expression during early porcine cleavage stages indicative of higher quality embryos (Yang et al. 2018). Significantly, disruption of mitochondrial fission via Drp1 siRNA results in chromosomal instability and over-amplification of centromeres in a number of cell lines (Qian et al. 2012), suggesting that the incidence of aneuploidy may reflect compromised mitochondrial dynamics, again linking mitochondrial function to chromosomal segregation. Additionally, ROS are also known to directly alter the epigenetic landscape (Hitchler & Domann 2009), such that conditions that result in the overproduction of ROS, including through the disruption of mitochondrial fission/fusion, may result in ROS-mediated changes to the epigenetic landscape. A second key component of mitochondrial fission is Fis1 (Fission 1), a transmembrane protein localised to the outer mitochondrial membrane, recruits Drp1 to areas of mitochondrial constriction, resulting in membrane fission (Stojanovski et al. 2004). However, no studies have addressed the role of Fis1 during mammalian embryo development.

Collectively, these data reveal that a balance between mitochondrial fission and fusion is required to regulate developmental progression and embryo viability, likely to ensure segregation of mitochondria between cells and ensure appropriate regulation of chromosome segregation, but also support appropriate mitochondrial activity and ROS levels. This balance can be perturbed by changes in nutrient availability, leading to mitochondrial fragmentation or hyperfusion and is dependent on mitochondrial membrane potential (Ishihara et al. 2003). Whether active fission and fusion events are altered in in vitro cultured embryos, or by alterations in nutrient availability, have not been examined. While knockout studies highlight the significance of regulators of mitochondrial dynamics during in vivo development, few studies have addressed changes that may be induced by loss of these proteins prior to implantation that may correlate with reduced potential or implantation failure. Equally, studies examining modulation of mitochondrial dynamics have not assessed whether changes in methylation or acetylation occur.

The dynamic nature of mitochondria: relocalisation to facilitate stage-specific epigenetic programming?

Mitochondrial localisation also changes significantly throughout oocyte and early embryo development. Given the role of mitochondrially derived metabolic intermediates in regulating the epigenome, mitochondrial trafficking may serve to not only transport mitochondria to sites of energy demand, and facilitate communication and interaction with other organelles to support embryo development, but also to maintain metabolite/signalling gradients with the nucleus.

Although subtle species differences exist, significant mitochondrial (re)distribution occurs in the developing oocyte (Takahashi et al. 2016), with distinct patterns of localisation in pronuclear oocytes and early cleavage stage embryos correlated with developmental potential (Wang et al. 2009c, Igosheva et al. 2010, Yu et al. 2010). In mouse and human oocytes, highly polarised mitochondria cluster around the oocyte cortex (Van Blerkom et al. 2003, Van Blerkom & Davis 2006, 2007), proposed to maintain sufficient ATP for fertilisation, but also to support Ca2+ regulation upon fertilisation. In contrast, mitochondria with low membrane potential are homogeneously distributed throughout the cytoplasm. The cortical localisation of active mitochondria may conceivably be required to support oocyte metabolism through close proximity of mitochondria to the source of pyruvate (the surrounding cumulus cells) given that the oocyte is unable to metabolise glucose (Sutton-McDowall et al. 2010) or establish nutrient gradients to modulate pronuclear dynamics. Localised ATP bursts have also been observed during mouse oocyte maturation, correlating with the redistribution of mitochondria (Yu et al. 2010), but may equally be indicative of fluctuations in other mitochondrially derived metabolites in a pulsatile manner.

Following fertilisation, mitochondria translocate to cluster around the two pronuclei (Motta et al. 2000, Squirrell et al. 2003). Mitochondrial translocation to a perinuclear arrangement has been observed in hamster (Barnett et al. 1996), mouse (Van Blerkom & Runner 1984), pig (Sun et al. 2001), rhesus macaque (Squirrell et al. 2003) and human (Noto et al. 1993, Van Blerkom et al. 2000) zygotes, providing ATP and other intermediates to facilitate syngamy and ensure an even organelle distribution between resultant blastomeres. Intriguingly, the relocation of mitochondria coincides with differential modulation of the paternal and maternal epigenomes, such that clustering likely establishes crosstalk between the nuclear and mitochondrial genomes, and may establish local metabolite domains to regulate epigenetic modifiers. Interestingly, nuclear expression of mitochondrial TCA enzymes, necessary to produce metabolites required for epigenetic remodelling at the time of EGA, has recently been described in mouse embryos (Nagaraj et al. 2017), and relies on pyruvate availability, indicating a need for temporal nutrient requirements to regulate the preimplantation epigenetic landscape. The dynamic changes to the epigenetic landscape that occur at syngamy may therefore necessitate translocation of mitochondria to the pronuclei to provide essential metabolites, including α-KG and NAD+ to support demethylase activity. Dumollard et al. (2007) reported that NAD(P)H levels significantly decreased following fertilisation, an inverse indicator of an increase in NAD+ availability, supporting this hypothesis. However, temporal changes in NAD(P)H levels during syngamy remains relatively unexplored. Significantly, a lack of mitochondrial organisation at this stage has been correlated with reduced implantation (Wang et al. 2009b), with mitochondrial aggregation characteristic of oocytes with reduced developmental potential (Igosheva et al. 2010), suggesting that these dynamic properties of mitochondria are essential for embryo development. Further, ovarian stimulation regimen has a substantial impact on mitochondrial distribution in the mouse (Ge et al. 2012) and human oocyte (Dell’Aquila et al. 2009), indicating that stimulation can significantly alter mitochondrial dynamics, which may have consequences for implantation, the induction of aneuploidies, viability and long-term health through the modulation of epigenetics.

Perturbations in mitochondrial movement and recruitment are also likely to lead to subcellular ATP depletion and interfere with mitochondrial Ca2+ signalling. Mitochondria play a role in Ca2+ storage and homeostasis (Duchen 2000), with sustained Ca2+ oscillations following fertilisation necessary for oocyte activation (Kline & Kline 1992) and embryo development (Stricker 1999). Mitochondrial localisation, and activity, interplays with Ca2+ signalling at fertilisation, such that mitochondrial ATP production is required for maintaining Ca2+ levels in the oocyte (Wakai et al. 2013) and for sustaining Ca2+ oscillations post-fertilisation (Dumollard et al. 2004). Significantly, mitochondrial movement along microtubule tracks is regulated by Ca2+ sensitive microtubule associated proteins, including kinesins, dyneins and myosins that drive motor-based movement (Varadi et al. 2004, Lawrence et al. 2016), suggesting that mitochondrial distribution relies on their own activity, as well as the integrity of the cytoskeletal network, to sustain localised signalling. Indeed, inhibition of microtubule polymerisation in human oocytes reduces germinal vesicle breakdown (GVBD) and prevents the completion of maturation, accompanied by the loss of mitochondrial redistribution (Takahashi et al. 2016), and cytoskeletal disruption in mouse eggs alters mitochondrial ATP production (Yu et al. 2010). Consequently, altered mitochondrial activity may reduce their ability to modulate Ca2+ dependent interactions with the cytoskeleton.

Connectivity and communication between mitochondria, the nucleus and other organelles, as well as the cytoskeleton, is therefore essential to the maintenance of cellular homeostasis in the oocyte and developing embryo, integrating diverse environmental signals as in other cell types (reviewed by Anesti & Scorrano 2006, Rowland & Voeltz 2012, Marchi et al. 2014) and eliciting signalling cascades that support preimplantation development. Loss, or misregulation, of these close interactions may therefore negatively impact the embryo’s ability to respond to stress and regulate mitochondrial function, or lead to precocious or delayed modification of the epigenetic landscape, particularly in response to changes in nutrient availability, thereby impacting long-term viability and health.

Limitations to assessing mitochondrial DNA content in isolation

While mitochondrial health and functionality relies on metabolic activity, distribution, morphology and density, an often-overlooked characteristic that separates mitochondria from other organelles is that each mitochondrion contains numerous copies of its own double stranded genome (mitochondrial DNA; mtDNA). mtDNA encodes 13 proteins involved in the respiratory chain, along with 22 transfer RNAs and 2 ribosomal RNAs (Anderson et al. 1981, Taanman 1999, Clayton 2000); however a significant proportion of genes required for mitochondrial biogenesis, homeostasis and regulation of OXPHOS are encoded by the nuclear genome. Coordinated signalling between the nucleus and mitochondrion is therefore essential to regulate mitochondrial function (reviewed elsewhere by St John et al. 2010, Harvey et al. 2011), and metabolic intermediate levels, and therefore the epigenome. Indeed, a role for mtDNA, and therefore mitochondrial activity, in regulating the nuclear epigenetic landscape is evidenced by a study examining mtDNA depletion which results in aberrant CpG methylation that can be reversed following repletion of mtDNA (Smiraglia et al. 2008). Thus, deficiencies in mtDNA may be paralleled by changes to the epigenome that reduce developmental competence.

The mature human and mouse oocyte contains an estimated 20,000 to 800,000 mtDNA copies (Steuerwald et al. 2000, Reynier et al. 2001, Barritt et al. 2002) and between 10,000 to 650,000 copies (Piko & Taylor 1987, Steuerwald et al. 2000, Aiken et al. 2008, Cree et al. 2008) respectively, with mtDNA levels remaining low following fertilisation, only increasing around the time of implantation (Thundathil et al. 2005, May-Panloup et al. 2005b, Bowles et al. 2007, Spikings et al. 2007, Aiken et al. 2008, Mtango et al. 2008). A threshold of 40, 000 to 50, 000 mtDNA copies in a mature mouse oocyte has been proposed as critical to support development post-implantation (Wai et al. 2010). While blastocysts still develop with as few as 4000 mtDNA copies, extrapolated from 8-cell embryos, those with low mtDNA levels failed to develop post-implantation (Wai et al. 2010). Likewise, while unfertilised human oocytes and their associated somatic cells harbour higher levels of mtDNA mutations and deletions (Keefe et al. 1995, Seifer et al. 2002, Hsieh et al. 2004), associated with reduced expression of mitochondrial genes (Hsieh et al. 2002), and oocytes from older females being more likely to possess deletions (Keefe et al. 1995, Jacobs et al. 2007, Hammond et al. 2016), how this relates to oocytes within the cohort that fertilise is difficult to determine. Significantly, levels vary considerably within one individual, and can be altered by ovarian stimulation regimen (Cree et al. 2015). Moreover, mtDNA copy number is often assumed to be an indicator of mitochondrial number, yet each mitochondrion can contain a varying number of mtDNA nucleoids (Gilkerson et al. 2013). Combined with species’ differences, and the use of discarded and poor-quality human samples, the utility and specificity of these thresholds is potentially limited.

Despite these limitations, mtDNA defects have been associated with ageing and the failure of mature oocytes to fertilise. mtDNA levels decline in oocytes with advancing maternal age (de Boar et al. 1999, Murakoshi et al. 2013) and unfertilised oocytes contain significantly fewer copies of mtDNA, suggesting that mtDNA copy number may be indicative of fertilisation potential (Reynier et al. 2001, Santos et al. 2006, Wai et al. 2010). Ovarian insufficiency has also been characterised by a significantly lower mtDNA content in oocytes (May-Panloup et al. 2005a), as well as accompanying cumulus cells (Boucret et al. 2015). Further, blastocysts from high fat diet (HFD) fed mice contain fewer mtDNA copy numbers and give rise to heavier foetuses in which both liver and kidney mtDNA content is significantly reduced (Wu et al. 2015), indicating that altered nutrition prior to conception can disrupt mitochondrial transmission. In addition to a lowered capacity for ATP generation, reduced mtDNA levels likely result in a diminished pool of TCA metabolites that are preferentially shunted toward ATP production to support cell survival in lieu of histone modification, predisposing the developing embryo to compensatory changes in physiology and growth. Indeed, mitochondrial DNA depletion has been shown to result in alterations in one-carbon metabolism (Bao et al. 2016) and TCA activity, altering transcription of genes involved in acetyl-CoA metabolism, and modifies amino acid metabolism, particularly that of methionine, consequently impacting on methylation (Lozoya et al. 2018). Conversely, enhanced mitochondrial OXPHOS capacity with increased mtDNA content may result in an increased availability of mitochondrial metabolites that results in hypermethylation and/or acetylation, likewise impacting downstream growth and physiology.

Accumulating evidence suggests that epigenetic changes also occur within mtDNA. While low levels (<5%) of mtDNA methylation were reported in mouse and human cells in the 1980s (Shmookler Reis & Goldstein 1983, Pollack et al. 1984), mitochondria were believed to lack histones, the major component of chromatin for epigenetic modification, such that the existence of mtDNA methylation was, and remains, controversial. While speculation remains around potential sample contamination, histones have been detected in the mitochondrial membrane (Choi et al. 2011). Combined with the identification of mtDNA methyltransferase enzyme 1 (mtDNMT1), associated with mitochondrial cytosine methylation (Shock et al. 2011) and the isolation of DNMT3a from mitochondria (Wong et al. 2013), it is possible that methylation actively takes place within mitochondria. In support of this, mitochondrial targeted expression of DNA methyltransferases is sufficient to induce mtDNA methylation (van der Wijst et al. 2017) and (de)methylation of the D-loop has been associated with a reduction in mtDNA copy number (Tong et al. 2017), which would reduce mitochondrial gene expression. The mtDNA methylation may consequently alter the availability of the co-factors necessary for the activity of nuclear epigenetic modifiers, through the modulation of mtDNA encoded genes and therefore OXPHOS activity, although direct evidence for this, and how mitochondrial methyltransferases are regulated, is currently lacking. Similarly, given that mitochondria, and mtDNA, are more susceptible to damage, and rely on cellular antioxidant systems to prevent damage, the mtDNA epigenome may be even more vulnerable than the nuclear epigenetic landscape. Consequently, it will be important to investigate the effects of not only culture, but other aspects of assisted reproductive technologies, particularly mitochondrial replacement, on the oocyte and embryo nuclear and mtDNA epigenome, particularly given the potential for altering offspring health.

Controversially, studies have emerged suggesting that a higher mtDNA copy number (e.g. Mitoscore >97; Diez-Juan et al. 2015), in blastomere or trophectoderm biopsies, or in cumulus cells, is an indicator of poor blastocyst quality and/or implantation success (Diez-Juan et al. 2015, Fragouli et al. 2015, Ogino et al. 2016, Desquiret-Dumas et al. 2017, Ravichandran et al. 2017), with the increase suggestive of underlying stress and compensatory mitochondrial biogenesis activated in an attempt to rescue development. This is however at odds with the metabolic requirements of the blastocyst stage embryo (Gardner & Harvey 2015). Moreover, these scores rely on an artificial reference threshold, and do not account for differences in mtDNA levels that exist between individual cells or cell types, which has been documented within the embryo (Cree et al. 2008). Indeed, mtDNA segregation has recently been shown to be non-random, rather relating to the level of OXPHOS and mitochondrial membrane potential (Otten et al. 2018), the activity of which differs considerably between the trophectoderm and the ICM (reviewed by Gardner & Harvey 2015). However, a direct determination of neither whether a consistent relationship exists in both the trophectoderm and ICM nor whether defective mitochondria are preferentially segregated into the trophectoderm relative to the ICM, or whether trophoblast biopsies presenting with higher mtDNA levels reflect a significant reduction in ICM levels that contribute to their demise has been established.

Conversely, studies have shown mtDNA content to have no correlation with implanting and non-implanting embryos (Treff et al. 2017, Victor et al. 2017), although significant mathematical correction may preclude the identification of a relationship. Further, there appears to be little predictive value at moderate (e.g. 75th percentile) mtDNA levels (Klimczak et al. 2018), with considerable range overlap apparent across all studies to date. Methodological differences between species and clinics, including but not limited to stimulation regimens and maternal age (Cree et al. 2015, Fragouli et al. 2015), developmental stage relating to cell number and volume and developmental kinetics (Ho et al. 2018), but also culture conditions that alter nutrient availability, likely impact the determination of mtDNA copy number, although oxygen concentration does not appear to be a factor (de Los Santos et al. 2018). Consequently, the utility of blastocyst mtDNA copy number as a biomarker of embryo viability, in isolation, is questionable and will result in embryos that would produce a viable pregnancy being classified as non-viable; a travesty for those with limited embryo numbers and opportunities to become pregnant. While the use of mtDNA copy number is likely to be somewhat informative, it presents an oversimplistic view of the potential significance of mitochondrial function as a biomarker, particularly in light of their role in mediating the epigenome. Rather, mitochondrial, and mtDNA, integrity, taking into account activity, content (both mitochondrial and mtDNA heteroplasmy and mutational load) and mitochondrial dynamics (including morphology and distribution) will need to be integrated, together with existing markers and epigenetic analyses, in order to be of predictive value and to establish a correlation with post-transfer outcomes.

Mitochondrial replacement therapy: proceed with caution

Mitochondrial replacement therapy (MRT), approved for use in the United Kingdom in 2015, has been proposed as a viable option to prevent the transmission of debilitating mitochondrial diseases. The technique involves the removal of donor nuclear material, and replacement with the diseased individual’s DNA, either via pronuclear, spindle or polar body transfer. However, the procedure is only applicable to cases where the mitochondrial disease is encoded by mtDNA, excluding the vast majority of cases in which mitochondrial disease is nuclear encoded. MRT pregnancies are currently ongoing, although controversially, the first baby born using the technique was reported in 2016 (Zhang et al. 2017).

However, the technology brings with it the potential for long-term health effects, particularly given the plethora of roles mitochondria have in regulating not only metabolism, but also localised signalling cascades and intracellular interactions as noted herein. Of further concern is the potential for the retention of diseased maternal mtDNA, resulting in heteroplasmy, which can remain undetected (Tachibana et al. 2013), or be detected at levels of up to 10% in foetal tissues (Zhang et al. 2017). Sustained heteroplasmy has been reported following cytoplasmic transfer into human oocytes (Brenner et al. 2000) with varying proportions of mtDNA observed in different tissues (Barritt et al. 2001), although whether the technique and/or heteroplasmy has directly affected offspring health and disease risk is unclear. Heteroplasmy has also been reported in cloned animals, with proportions ranging from 0.5 to 40% (Hiendleder et al. 1999, Takeda et al. 1999, 2003, Steinborn et al. 2000, Hiendleder et al. 2003), although these figures are likely underestimates given the lack of sensitivity of the techniques used (Hammond et al. 2016). Ma et al. (2015) reported that mitochondrial deficits could be corrected with somatic cell nuclear transfer and genetic manipulation, however varying (0–100%) levels of heteroplasmy remained detectable in these lines. While heteroplasmy can occur in normal individuals, and varies across tissues, it has been associated with mitochondrial disease and metabolic perturbations in mice (Acton et al. 2007, Sharpley et al. 2012) with severity depending on the threshold level of diseased mitochondria. Significantly, a disease phenotype can occur even at very low (<5%) levels of heteroplasmy for certain deficits (Chinnery et al. 1997, Sacconi et al. 2008). While the birth of live macaque offspring (Tachibana et al. 2009) facilitated the development and adoption of this technique, longitudinal, whole systems studies of the health of resultant offspring is warranted, combined with care to ensure the absence of mitochondrial carry over from the patient.

Beyond the potential for heteroplasmy, the technique neglects the requirement for coordinated communication between mitochondria and the nucleus, along with the dynamic regulation of mitochondrial movement during oocyte maturation and fertilisation. As proposed, the recruitment of mitochondria to the pronuclei preceding syngamy (Barnett et al. 1996, Squirrell et al. 2003) may be required to modulate the local metabolite environment to facilitate syngamy, subsequent cytokinesis and differentially modify the parental genomes, a process that may well be initiated prior to pronuclear migration. Further, given the significance of microtubules to mitochondrial distribution and chromosome segregation as noted above, it is therefore likely that a loss in microtubule network integrity will impact on further development via impairment of mitochondrial trafficking. Mitochondrial distribution following cytoplasmic transfer has been shown to depend on oocyte stage (Fulka 2004), possibly reflecting stage-specific changes in microtubule dynamics. Further, asymmetrical mitochondrial distribution at the pronuclear stage results in blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity (Van Blerkom et al. 2000), and plausibly altered paternal/maternal methylation characteristics that contribute to the observed reduction in developmental competence. Therefore, studies will need to address whether MRT methodologies alter mitochondrial and cellular metabolic regulation, as well as the embryo transcriptome and epigenome, to ensure minimal risk to resultant offspring health and the absence of transgenerational effects.

Conclusions

The involvement of mitochondria in regulating oocyte and embryo development goes beyond their role in generating ATP to support energy demanding processes. It is increasingly recognised that mitochondria are dynamic signalling organelles, trafficking to specific regions within the cell and communicating not only with the nucleus but also other organelles; interactions that facilitate signalling cascades and impact chromosome segregation, cytokinesis and lineage specification. Loss of mitochondrial function, including activity, organelle interactions and trafficking, and the mechanisms that modulate mitochondrial and mtDNA integrity, manifests as reduced development, and can result in failure to develop post-implantation. Combined with our evolving understanding of how metabolite availability and metabolic and mitochondrial pathway flux can drastically affect the availability of co-factors required for the activity of epigenetic modifiers, mitochondria are emerging as sensors that integrate not only metabolic and cell signalling cues but may in part explain the origins of poor developmental outcomes through heritable changes that lead to disease susceptibility. Therefore, while the developing embryo exhibits a degree of plasticity to changes in its extracellular environment, enabling development to the blastocyst stage to continue, it is nonetheless uniquely sensitive to perturbations in nutrient availability at a time when the epigenetic landscape is being established. The complexity of their diverse roles is of particular significance given the adoption of assisted reproductive technologies that detect or manipulate mitochondria or mtDNA numbers and potentially disrupt mitochondrial-organelle and -cytoskeletal communication and trafficking. This complexity necessitates the determination of mitochondrial characteristics in combination with epigenetic change in order to provide a more accurate understanding of the impact of mitochondrial deficits and manipulation on oocyte, embryo and offspring health, as well as their utility as biomarkers.

Declaration of interest

The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Acknowledgements

The author would like to thank Professor David K Gardner and Dr Mark P Green for their valuable comments on the manuscript.

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