Development of aerobic metabolism in utero : requirement for mitochondrial function during embryonic and foetal periods

Introduction The mammalian embryo requires an increasing amount of energy as it develops during the embryonic and foetal stages. The main production mechanism of adenosine triphosphate (ATP) during the early developmental period (post-implantation to beginning of organogenesis) is via anaerobic glycolysis. Despite the fact that it has long been known that embryonic/foetal development becomes dependent upon oxidative phosphorylation in mitochondria, a common misconception persists in the scientific literature indicating that a shift to aerobic metabolism occurs only after birth (‘foetal-shift’). Biotechnology has facilitated the creation of targeted genetic knockout models in mice, and many of these clearly demonstrate the necessity of mitochondrial function for proper development of the embryo/ foetus in the womb. This review highlights representative examples where the loss of genes influencing mitochondrial structure and function causes severe energy-deficiency and/or mitochondrial dysfunction leading to prenatal lethality. Conclusion Critical examination of the idea that anaerobic metabolism is the primary generator of ATP throughout gestation in mammals suggests that this may not be entirely true since the developmental switch to aerobic metabolism actually occurs well before birth. This earlier ‘embryonic-shift’ in metabolic mechanism is essential for embryonic survival and foetal development that begins during organogenesis and continues throughout the remainder of prenatal development. Introduction The primary source of adenosine triphosphate (ATP) during foetal development is commonly reported to be from glycolysis and lactate production, with a ‘foetal-shift’ occurring at birth where the primary source of ATP production ‘shifts’ to oxidative phosphorylation in the mitochondria1-3. A typical example of this notion from the literature is stated as follows1,3: ‘...metabolic programming often is referred to as a ‘foetal’ shift because the myocardium of the developing embryo relies mostly on glycolysis and lactate metabolism for its ATP production’. It has been known for more than 40 years, however, that mammalian hearts show an increase in the importance of oxidative phosphorylation for ATP production during the embryonic development4-6. Additionally, there is accumulating evidence from targeted genetic studies in mice showing embryonic lethality due to disruption of genes associated with the mitochondria and/or dysfunctional oxidative phosphorylation. Moreover, cardiovascular development has been well studied using transgenic mice demonstrating embryonic lethality occurring around the time of mitochondrial maturation during organogenesis7,8. Organogenesis begins around embryonic day 8.0 (E8.0) in mice, which is equivalent to about day 17–19 (Carnegie Stage 8) in humans9,10. Interestingly, this is approximately the stage of development when increased embryonic lethality due to the genetic disruption of mitochondrial associated genes occurs (Table 1) thereby demonstrating that embryonic mitochondrial function is indeed critical for embryonic and foetal development in utero. The aim of this review was to discuss the requirement for mitochondrial function during embryonic and foetal periods in the development of aerobic metabolism in utero. Genetic knockouts of mitochondrial DNA-associated genes Mitochondria are unique organelles as they contain their own DNA (mtDNA) encoding for a set of 37 mitochondrial genes. These genes encode for a variety of proteins including ribosomal RNAs, transfer RNAs and 13 subunits of the electron transport chain11. Although mitochondria contain their own DNA, the majority of the proteins found within the mitochondria are encoded by nuclear DNA. Nuclear DNA encodes all proteins required for mtDNA synthesis. The only DNA polymerase found within mitochondria, DNA polymerase gamma (Polg) is thought to be solely responsible for the replication and repair of mtDNA12. Mutations of the mouse Pol I-like catalytic core (PolgA) gene resulted in an embryonic lethal model that died between E7.5–E8.5 with severe mtDNA deletions13. Additionally, this model demonstrated a respiratory chain dysfunction by a lack of cytochrome c oxidase (COX) staining13. A similar model with the disruption of a Polg accessory subunit, Polg2, resulted in lethality between E8.0–E8.5 due to defective * Corresponding author Email: steven.ebert@ucf.edu 1 Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida 32827, USA Ph ys io lo gy & B io ch em is tr y


Introduction
The primary source of adenosine triphosphate (ATP) during foetal development is commonly reported to be from glycolysis and lactate production, with a 'foetal-shift' occurring at birth where the primary source of ATP production 'shifts' to oxidative phosphorylation in the mitochondria [1][2][3] .A typical example of this notion from the literature is stated as follows 1,3 : '…metabolic programming often is referred to as a 'foetal' shift because the myocardium of the developing embryo relies mostly on glycolysis and lactate metabolism for its ATP production'.It has been known for more than 40 years, however, that mammalian hearts show an increase in the importance of oxidative phosphorylation for ATP production during the embryonic development [4][5][6] .Additionally, there is accumulating evidence from targeted genetic studies in mice showing embryonic lethality due to disruption of genes associated with the mitochondria and/or dysfunctional oxidative phosphorylation.Moreover, cardiovascular development has been well studied using transgenic mice demonstrating embryonic lethality occurring around the time of mitochondrial maturation during organogenesis 7,8 .Organogenesis begins around embryonic day 8.0 (E8.0) in mice, which is equivalent to about day 17-19 (Carnegie Stage 8) in humans 9,10 .
Interestingly, this is approximately the stage of development when increased embryonic lethality due to the genetic disruption of mitochondrial associated genes occurs (Table 1) thereby demonstrating that embryonic mitochondrial function is indeed critical for embryonic and foetal development in utero.The aim of this review was to discuss the requirement for mitochondrial function during embryonic and foetal periods in the development of aerobic metabolism in utero.

Genetic knockouts of mitochondrial DNA-associated genes
Mitochondria are unique organelles as they contain their own DNA (mtDNA) encoding for a set of 37 mitochondrial genes.These genes encode for a variety of proteins including ribosomal RNAs, transfer RNAs and 13 subunits of the electron transport chain 11 .Although mitochondria contain their own DNA, the majority of the proteins found within the mitochondria are encoded by nuclear DNA.Nuclear DNA encodes all proteins required for mtDNA synthesis.The only DNA polymerase found within mitochondria, DNA polymerase gamma (Polg) is thought to be solely responsible for the replication and repair of mtDNA 12 .Mutations of the mouse Pol I-like catalytic core (PolgA) gene resulted in an embryonic lethal model that died between E7.5-E8.5 with severe mtDNA deletions 13 .Additionally, this model demonstrated a respiratory chain dysfunction by a lack of cytochrome c oxidase (COX) staining 13 .A similar model with the disruption of a Polg accessory subunit, Polg2, resulted in lethality between E8.0-E8.5 due to defective transcription and is implicated to have a role in mitochondrial genome replication 15 .The ablation of Tfam produced an embryonic lethal mouse model with attrition occurring between E8.5 and E10.5 16 .A decrease of COX staining, enlarged mitochondria with disorganisation of cristae and increased apoptosis indicates lethality was due to impairment in respiratory chain function 16 .Ribonucleases H (RNases H) have been associated with mtDNA replication by removal of RNA from RNA-DNA hybrids or primers of Okazaki fragments 17 .Cerritelli et al. 17 showed genetic ablation of RNaseH1, which resulted in a drastic loss of mtDNA and, like the Tfam -/-, displayed an increase in apoptosis and embryonic lethality beginning at E8.5.Additionally, the mitochondrial and nuclear localisation of this ribonuclease was shown through co-localisation experiments in human (HeLa) and monkey (Cos-1) cell lines 17 .While mechanisms are currently understood about the initiation of mtDNA transcription, little is known regarding regulation of mtDNA transcriptional.Mammalian mitochondrial transcription termination factor 3 (Mterf3) is a negative regulatory protein that has been identified in correlation with mtDNA transcription 18 .Mterf3 is essential for embryonic development past E8.5 18 .Tissue-specific deletion of Mterf3 in the heart and skeletal muscle causes mitochondrial dysfunction, though specific characterisation in embryonic tissue has not yet been performed 18 .
The majority of mitochondrial proteins are translated via cytosolic ribosomes and then translocated through membrane pores in the mitochondria.Relatively little is known regarding the mechanistic details of mtDNA translation.The translational protein p32 was found to localise to the mitochondria and associate with transcriptional protein Tfam 19 .p32-deficient mice were severely underdeveloped and died during midgestation (E10.5-E11.5)due to impaired oxidative phosphorylation 20 .p32 -/- mouse embryonic fibroblasts (MEFs) displayed characteristic mitochondrial respiratory dysfunction, including abnormal mitochondrial morphology, decreased complex I, II and IV activity, impaired ATP generation and reduced mitochondrial membrane potential (Δψm) 20 .

Genetic knockouts of mitochondrial genes associated with fi ssion and fusion
Mitochondria are dynamic organelles that are constantly undergoing fission and fusion 21 .The primary machinery involved in fission is the dynamin-related protein1 (Drp1), while mitochondrial fusion is facilitated by mitofusins (Mfn) 1 and 2 and optic atrophy 1 (Opa1) [22][23][24] .6][27] .The loss of Drp1 results in growth retardation and attrition after E10.5, likely due to defective mitochondrial division as shown by electron micrographs of elongated mitochondria in Drp1 -/-MEFs; however, intracellular ATP levels were not altered in the knockouts as compared to the wild-types 25 .Mfn1 and Mfn2 have each been individually genetically disrupted, with slightly different resulting characteristics.Mfn1 -/-embryos die beginning at E10.5 while Mfn2 -/-embryos have increased reabsorption beginning at E9.5 with observed placental defects not present in Mfn1 -/-embryos.Both models demonstrated fragmented spherical mitochondria and altered dynamics resulting in lethality 28 .Recently, a cardiac-specific knockout model of both Mfn1/2 was generated yielding a similar embryonic lethal phenotype at E9.5 with comparable mitochondrial morphologic defects 26 .Finally, the disruption of Opa1 expression results in embryonic lethality before E11.0 in homozygous mutants, though complete characterisation of this model has not yet been described 27 .

Genetic knockouts of electron transport chain-associated genes
The electron transport chain is the main mechanism of ATP production in the postnatal period, and it is located within the inner mitochondrial membrane.It is composed of five complexes (I-V) each with distinct subunits, and is a significant generator of harmful reactive oxygen species (ROS) during respiration (Figure 1).The disruption of the D subunit of succinate dehydrogenase (SDH), nuclear-encoded and located within complex II, results in early embryonic lethality ranging from E6.5-E7.5, speculated to be a result of increased energy demands 29 .Cytochrome c (Cytc), a mitochondrial electron transport carrier and 'initiator' of caspase-mediated apoptosis, was shown to be developmentally necessary after E8.5 30 .Complex IV of the electron transport chain, also known as COX, converts oxygen to water using Cytc as the electron donor.Ablation of the synthesis of cytochrome c oxidase (Sco2) gene, a COX assembly protein, results in embryonic lethality around E8.5 showing decreased COX and SDH activities 31 .
As mentioned above, active electron transport chain machinery is a large producer of ROS.While production of ROS is necessary for development, regulation of ROS accumulation is vital as well.The disruption of ROS regulatory factors, thioredoxin 2 (Trx-2) and thioredoxin reductase (TrxR2) each result in midgestational lethality at E10.5 and E13.0, respectively 32,33 .As expected, an increase in ROS was observed in both models; however, thinning of the ventricular wall and decreased haematopoiesis were also observed in the TrxR2 -/- embryos 33 .

Genetic knockouts of mitochondrial biogenesis genes
Mitochondrial biogenesis requires a responsive network of genes that have many sensory as well as effector targets.Peroxisome proliferator-activated receptor γ (Pparγ) has been implicated in the initiation of mitochondrial biogenesis 34 .Cardiacspecific deletion of Pparγ leads to increased oxidative damage and cardiac hypertrophy 34 .Global genetic deletion of Pparγ produced an embryonic lethal phenotype around E10.0, associated with abnormal mitochondria, ventricular wall thinning and placental vascularisation abnormalities 35 .Major players in mitochondrial biogenesis are the Pparγ coactivators-1α and -1β (Pgc-1α and Pgc-1β).The combinatorial knock-out of these genes results in lethality at postnatal day 1 36 .These mice exhibit symptoms of cardiac failure associated with the expected block in mitochondrial biogenesis 36 .

Genetic knockouts with dysfunctional mitochondria
While it is expected that the deficiency of mitochondrial-associated genes would cause a disturbance of oxidative phosphorylation, numerous murine models have been created that exhibit a loss of mitochondrial function as a result of seemingly non-associated genetic knockouts.For example, the nuclear factor of the activated T-cell (NFAT) family of transcription factors are generally associated with immune responses and cardiovascular development 37 .The disruption of nfatc3 and nfatc4 in combination leads to embryonic lethality between E10.5-E11.0due to cardiac failure 38,39 .Mitochondrial function was evaluated by COX and SDH staining and revealed decreased activity of both, as well as electron micrographs with swollen mitochondria with disordered cristae in nfatc3 -/-nfatc4 -/-embryos 38 .Nfatc4 individual knockout mice showed no phenotype upon 36 months of observation; however, nfatc3 knockout mice displayed impaired T cell function with some prenatal lethality (approximately 50% of nfatc3 -/-die in utero) 39,40 .
Another example is the parathyroid hormone type 1 receptor (Pth1r), which is expressed in multiple tissue types throughout the body.Disruption of the Pth1r gene results in embryonic lethality beginning at E12.0, with a similar phenotype as the nfatc3 -/-nfatc4 -/-model, including cardiac failure and altered ultrastructure of embryonic mitochondria 39,41 .Another interesting case of midgestational lethality is due to metabolic defects resulting from disruption of the retinoid x receptor alpha (RXRα -/-) gene 42 .These RXRα mutants have complete attrition of embryos due to energy deficiency by E13.5, with significantly lowered ATP concentrations and decreased complex I levels 42 .It is noteworthy to mention that the retinoid x receptor has been shown to form a heterodimer with Pparγ (knockout lethal at E10.0) to facilitate transcriptional regulation of specific target genes 43 .
Other genes that are known to be associated with mitochondria are the death-associated protein-3 (Dap3) and phosphatidylethanolamine decarboxylase (Psid) genes 44,45 .Dap3 has only recently been shown to localise to the mitochondria, and Dap3 -/-embryos revealed a lethal phenotype at E9.5 due to mitochondria that were approximately two-fold smaller than Dap3 +/+ littermates 44 .Lastly, the disruption of Psid, a phospholipid known to localise to the mitochondria, results in lethality between E8.0-E10.0 with fragmented mitochondria 45 .

Discussion
The disruption of several different mitochondrial-associated genes results in embryonic lethality during the critical organogenesis phase as listed in Table 1 and shown schematically in Figure 1.In support of this genetic evidence, electron microscopy has shown an obvious maturation of mitochondria and cristae development between E10-12 in rat embryos (equivalent to E9.0-E10.0 in mice) 46 .Further, the inhibition of glycolysis using the drug E9.0 mouse ATP concentrations, indicating an alternative mechanism of energy production (e.g.oxidative phosphorylation) 47 .Until recently, the study of oxidative phosphorylation and mitochondrial maturation in utero had not been thoroughly investigated, but a new mechanism for the induction of mitochondrial maturation was proposed involving the closure of the mitochondrial permeability transition pore (mPTP) beginning at E9.5 in mouse cardiomyocytes 48 .The closure of the mPTP led to increased oxidative phosphorylation and decreased ROS production in the E9.5 mouse heart, and has thus been proposed as a key mechanism in the maturation of mitochondrial function in the developing embryo at a time when the embryo becomes dependent on aerobic metabolism 48 .
Despite this clear and growing body of evidence, it is still often reported that the primary mechanism of ATP production during this developmental period occurs through anaerobic glycolysis.Part of the confusion appears to stem from the fact that there is a well-established shift in metabolic substrate utilisation by the heart after birth.Even though glucose is the predominant substrate during the prenatal period 6,49 , there is a shift (also referred to as a 'foetal-shift') towards utilisation of free fatty acids by the heart in the postnatal period, and this continues to be the primary metabolic substrate for cardiac function throughout adulthood 50 .It is nevertheless important to note that early in vitro studies have shown the necessity of oxygen, as well as glucose and lactate, to avoid metabolic dysfunction in isolated embryonic rat hearts and embryos 5,51 .Thus, while glycolysis remains the primary generator of 3-carbon substrates for the Kreb's Cycle throughout the prenatal period, the embryo and subsequent foetus cannot survive without activation of mitochondrial function and oxidative phosphorylation during the embryonic period.This idea which includes an 'embryonic-shift' from primarily anaerobic to aerobic metabolism is schematically illustrated in Figure 2.

Conclusion
Although this is not a comprehensive evaluation of all evidence and models relating to metabolic function in utero, we have highlighted several pertinent examples of genetic mutations that affect mitochondrial function and are necessary for embryonic development (Table 1 and Figure 1).These reports indicate a functionally important role for mitochondria during early stages of embryonic and foetal development in mammals.The ablation of genes associated with mitochondrial biogenesis and function causes attrition of embryos during critical stages of development in utero.Hence, oxidative phosphorylation clearly plays a pivotal role in ATP production during embryonic and foetal development.This does not in any way negate or conflict with a subsequent further shift to aerobic metabolism at birth, but does help to clarify some of the confusion currently in the literature about the relative roles of glycolysis versus mitochondrial oxidative phosphorylation during prenatal development.

Figure 1 :
Figure 1: Schematic representation of the cellular localisation of target genes Genes targeted for mutation have a variety of subcellular localisation, not all specifically in the mitochondria.mtDNA, mitochondrial DNA.

Figure 2 :
Figure 2: Timeline of attrition in genetic knock-out models that affect mitochondrial function ' X' represents a model of mitochondrial-associated lethality; vertical dashed lines indicate timing of 'embryonic-shift' from anaerobic to aerobic metabolism.Please note that arrows depicting timelines of activity for 'glycolysis' and 'oxidative phosphorylation' are qualitative and mainly serve to indicate the relative importance of glycolysis (and subsequent lactic acid formation through anaerobic means) and oxidative phosphorylation in mitochondria for proper embryonic and foetal development.

Table 1 . Mitochondrial and related genes essen al for development. Gene Age of death Phenotype Cita on mtDNA associated genes
Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) F : Baker CN, Ebert SN.Development of aerobic metabolism in utero: requirement for mitochondrial function during embryonic and foetal periods.OA Biotechnology 2013 Apr 01;2(2):16.
2-deoxyglucose did not show a decrease in Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) Development of aerobic metabolism in utero: requirement for mitochondrial function during embryonic and foetal periods.OA Biotechnology 2013 Apr 01;2(2):16.