CSIRO PUBLISHING

Review

Reproduction, Fertility and Development, 2015, 27, 572–582 http://dx.doi.org/10.1071/RD14343

Nutrient pathways regulating the nuclear maturation of mammalian oocytes Stephen M. Downs Department of Biological Sciences, Marquette University, 530 N 15 St, Milwaukee, WI 53233, USA. Email: [email protected]

Abstract. Oocyte maturation is defined as that phase of development whereby a fully grown oocyte reinitiates meiotic maturation, completes one meiotic division with extrusion of a polar body, then arrests at MII until fertilisation. Completion of maturation depends on many different factors, not the least of which is the proper provision of energy substrates to fuel the process. Interaction of the oocyte and somatic compartment of the follicle is critical and involves numerous signals exchanged between the two cell types in both directions. One of the prominent functions of the cumulus cells is the channelling of metabolites and nutrients to the oocyte to help stimulate germinal vesicle breakdown and direct development to MII. This entails the careful integration and coordination of numerous metabolic pathways, as well as oocyte paracrine signals that direct certain aspects of cumulus cell metabolism. These forces collaborate to produce a mature oocyte that, along with accompanying physiological changes called cytoplasmic maturation, which impart subsequent developmental competence to the oocyte, can be fertilised and develop to term. This review focuses on nuclear maturation and the metabolic interplay that regulates it, with special emphasis on data generated in the mouse. Additional keywords: AMP-activated protein kinase, energy substrates, fatty acid oxidation, germinal vesicle breakdown. Received 15 September 2014, accepted 10 January 2015, published online 24 March 2015 Introduction Oocyte nuclear maturation, defined as the meiotic progression from prophase I to MII, is a physiological event that precedes, and is required for, successful fertilisation and embryo development. Essential for meiotic maturation is the procurement of sufficient energy to fuel the myriad metabolic pathways that participate in the meiotic process. To accomplish this feat, a host of different factors must be carefully coordinated, including local and systemic signalling and regulatory give-and-take between germ cells and somatic tissues, all involving contributions from an intricate metabolic network. This review addresses different aspects of energy metabolism and how they interact to impact the nuclear maturation of mammalian oocytes, with particular emphasis on data generated in the mouse. For other aspects of oocyte maturation and developmental competence not covered in this review, the reader is directed to numerous excellent articles (e.g. Gilchrist and Thompson 2007; Sturmey and Leese 2008; Sturmey et al. 2009; SuttonMcDowall et al. 2010; Krisher et al. 2007). It is important to define three terms that will be used liberally throughout this review: (1) a ‘denuded oocyte’ (DO) is a naked oocyte that has had its surrounding cumulus cells removed; (2) a ‘cumulus cell-enclosed oocyte’ (CEO) is an oocyte still coupled to the cumulus cells (CEOs often behave differently to DOs because of the ability of the accompanying cumulus cells to influence oocyte metabolism and meiosis); and (3) the Journal compilation Ó CSIRO 2015

‘cumulus–oocyte complex’ (COC) is defined as the tissue unit containing both the oocyte and cumulus mass and is often used to refer to the collective metabolic response contributed by both cell types. The COC differs from CEO in that the latter refers specifically to the oocyte that happens to still be coupled to the cumulus cells. Energy substrates and spontaneous oocyte maturation When fully grown, meiotically competent oocytes are removed from their preovulatory follicles and placed in a suitable culture medium, they spontaneously undergo germinal vesicle breakdown (GVBD), extrude a polar body and develop to the MII stage. The energy substrates able to support this process have been determined for many species. A seminal paper by Biggers et al. (1967) showed that glucose and pyruvate are two principal carbohydrates that subserve this developmental event in mouse oocytes; their contributions to meiotic maturation are highlighted in Table 1. Both carbohydrates are readily available in the follicular fluid (Harris et al. 2005). The effectiveness of these substrates depends on the cell types present: whereas pyruvate is directly used by the oocyte and supports maturation of both DOs and CEOs, glucose is effective only in CEOs. This is because oocytes metabolise glucose very poorly (Biggers et al. (1967); Rushmer and Brinster 1973; Zuelke and Brackett 1992; Rieger and Loskutoff www.publish.csiro.au/journals/rfd

Energy and oocyte maturation

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Table 1. Effects of glucose and pyruvate on mouse oocyte maturation and related physiology PPP, pentose phosphate pathway; COCs, cumulus–oocyte complexes; DO, denuded oocyte Energy Substrate

Condition and/or effect

References

Glucose

Required for hormone-induced maturation via the PPP and de novo purine synthesis

Fagbohun and Downs (1992), Downs and Mastropolo (1994), Downs (1997a), Downs and Mastropolo (1997), Downs et al. (1998), Downs and Utecht (1999) Downs and Hudson (2000)

Pyruvate

Promotes optimal spontaneous maturation in combination with pyruvate (7) Promotes meiotic arrest via glycolysis-dependent increase in cumulus cell ATP that is delivered to the oocyte through gap junctions Ovulated (MII) COCs have higher rates of glucose metabolism than MI COCs Of negligible importance in DO maturation Acts cooperatively with amino acids to promote synthesis of purines involved in meiotic regulation Most important energy source for the oocyte Utilisation by oocytes correlates with meiotic activity More pyruvate is consumed by COCs but more is oxidised by DOs, implying meiosis-inducing action of pyruvate is not mediated by its oxidation Promotes maturation and is required for completion of maturation

1994; Downs and Utecht 1999), instead depending on the cumulus granulosa cells that efficiently convert glucose to pyruvate via glycolysis that they are able to use (Donahue and Stern 1968; Eppig 1976; Leese and Barton 1984). High consumption rates of pyruvate have been demonstrated in maturing cow (Rieger and Loskutoff 1994; Steeves and Gardner 1999), cat (Spindler et al. 2000), monkey (Brinster 1971) and human (Roberts et al. 2002) oocytes. Zuelke and Brackett (1992) reported that oocytectomised bovine complexes, in which the oocyte cytoplasm is removed leaving the cumulus oophorus intact, have a significantly repressed rate of glycolysis in response to LH. It was later determined in the mouse that oocyte-secreted growth factors upregulate glycolytic enzymes in the cumulus cells, thus enabling the oocyte to tap into the metabolic potential of these cells to produce pyruvate for their use (Sugiura et al. 2005, 2007). Indeed, glycolytic activity in oocytectomised mouse complexes is one-tenth that in intact complexes. Interestingly, the oocyte-secreted factor, recombinant human bone morphogenetic protein 15 (rhBMP15) , does not augment carbohydrate metabolism in bovine cumulus cells, but rather acts through the cumulus cells to increase oocyte oxidative phosphorylation (Sutton-McDowall et al. 2012). Of note, significant differences exist in the pattern of oocyte-secreted factors between species (Crawford and McNatty 2012). These studies serve to emphasise a central theme in cumulus cell function, which is to produce metabolites and nutrients for use by the oocyte (Cetica et al. 2002) that benefits by improved developmental competence (Gilchrist and Thompson 2007). Pyruvate-supported oocyte maturation depends on oxygen consumption via mitochondrial oxidative phosphorylation and generation of ATP (Haidri et al. 1971; Zeilmaker and Verhamme 1974; Magnusson et al. 1977). Lactate and

Downs and Mastropolo (1994), Downs (1995)

Harris et al. (2007) Downs and Utecht (1999) Downs and Verhoeven (2003), Downs (1998) Biggers et al. (1967) Harris et al. (2007), Downs et al. (1997, 2002a) Downs et al. (1997, 2002a)

Johnson et al. (2007)

oxaloacetate, both capable of conversion to pyruvate, also support maturation. Moreover, pyruvate has been shown to be important for both the initiation and completion of maturation (Johnson et al. 2007). Indeed, its consumption is greatest when the oocyte is actively engaged in meiosis and lowest at germinal vesicle and MII stages, whereas glucose consumption is high in meiotically arrested oocytes (Downs et al. 2002a); therefore, pyruvate consumption is positively correlated with the maturation process. A study with human oocytes also showed changes in pyruvate consumption according to meiotic status (Roberts et al. 2002). Although mouse COCs consume considerably more pyruvate than DOs, the latter are much more active in its oxidation (Downs et al. 2002a), suggesting that pyruvate consumption in cumulus cells may contribute to oocyte maturation beyond providing substrate for oxidation, consistent with alternative pathways for pyruvate metabolism. Bovine oocytes are apparently also more efficient than cumulus cells in pyruvate oxidation (Sutton-McDowall et al. 2012). However, species differences exist, because pyruvate does not support maturation in rat or rabbit oocytes (Kane 1972; Zeilmaker and Verhamme 1974; Bae and Foote 1975), and its consumption is inconsequential in maturing dog oocytes (Songsasen et al. 2007). Pyruvate supports spontaneous maturation of CEOs to the MII stage when culture is in a large (1 mL) volume of medium, but the presence of pyruvate alone is not sufficient to optimise this maturation. Instead, both pyruvate and glucose are required to maximise development to the MII stage (Downs and Hudson 2000; Songsasen et al. 2002; Fig. 1). Further, adequate levels of glucose are essential not only for nuclear maturation, but also for maximising the cytoplasmic maturation necessary for fertilisation and embryo development (Rieger and Loskutoff 1994; Steeves and Gardner 1999; Spindler et al. 2000; SuttonMcDowall et al. 2005, 2010; Herrick et al. 2006; Wongsrikeao

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100 90 80

Percent MII

70 60 50 40 30 20 10 0 [Pyr]/[Glc]: 0/0

0.23/0

1/0

0/5.5

0/27.8 0.23/5.5

1/5.5

Fig. 1. Effects of energy substrates on the completion of mouse cumulus cell-enclosed oocyte (CEO) maturation. CEOs were cultured for 15 h in minimum essential medium (MEM) containing varying combinations of pyruvate (Pyr) and glucose (Glc) and assessed for polar body formation (MII). Concentrations are in mM.

et al. 2006). Because cumulus cells can produce significant pyruvate from glucose and accumulation of pyruvate can be accentuated by culture in small volumes, high levels of maturation to MII can be achieved with glucose alone in microdrop (8 mL) cultures (Downs and Hudson 2000). Nevertheless, it is important to bear in mind that culture in small volumes, sometimes used to concentrate positive paracrine factors, could also have the unintended consequence of depleting important substrates that may be limiting under such conditions. Maintenance of meiotic arrest The spontaneous maturation of preovulatory oocytes outside the follicular environment indicates the somatic compartment of the follicle prevents premature meiotic resumption. One of the principal molecules contributing to this meiotic arrest is cAMP, which suppresses the activation of maturation promoting factor, the kinase responsible for driving the cell cycle. Thus, exposing cultured oocytes to cAMP-elevating agents such as cAMP analogues and cAMP phosphodiesterase inhibitors helps maintain them in meiotic arrest (for reviews, see Downs 2010; Jaffe and Norris 2010). However, the efficiency of this meiotic arrest in vitro is greatly influenced by the selection of culture medium and components within. Altering the relative levels of pyruvate and glucose in culture medium has a profound influence on whether oocyte maturation is suppressed or promoted. In the absence of glucose, pyruvate prevents the meiosis-suppressing action of meiotic inhibitors in mouse CEOs, whereas the reintroduction of only a small amount of glucose restores its inhibitory capacity (Fagbohun and Downs 1992; Downs and Mastropolo 1994; Downs 1995). The positive action of pyruvate does not depend on increased respiratory activity, because the substrate had no effect on ATP levels; conversely, the suppressive action of glucose on maturation is mediated by glycolysis-dependent ATP production. Arrest is accomplished by the diffusion of nucleotide to the oocyte

through heterologous gap junctions that metabolically couple the two cell types. Interestingly, metabolites of glucose analogues such as 2-deoxyglucose-6-phosphate also transit these gap junctions (Saito et al. 1994). We originally assumed that meiotic inhibition was achieved through the conversion of ATP to cAMP, but more recent work in our laboratory has suggested an additional possibility (see below). An important molecule involved in meiotic regulation of mouse oocytes is AMP-activated protein kinase (AMPK), an energy-sensing enzyme and metabolic regulator (Hardie et al. 2012; Steinberg and Kemp 2009). We have shown that AMPK has a positive effect on meiotic maturation, stimulating GVBD and meiotic progression to MII, as well as suppressing premature oocyte activation (Downs et al. 2002b, 2010; Chen et al. 2006; Chen and Downs 2008; Ya and Downs 2013). As is discussed later, fatty acid oxidation (FAO) plays a major role in AMPK action. AMPK activity is controlled by the AMP : ATP ratio: the higher this ratio, the greater the enzyme activity, leading to conserved energy use and increased energy production. I propose that increased oocyte ATP levels generated in the cumulus cells from glucose metabolism have a negative regulatory function in the mouse, entering the oocyte and dampening AMPK activity, thereby blocking its meiosis-inducing capacity. This action may be unique in the mouse, because it is the only species thus far tested where oocyte maturation is under positive control by AMPK. Indeed, stimulation of AMPK in cow, porcine and rat oocytes does not trigger meiotic resumption (Bilodeau-Goeseels et al. 2007; Mayes et al. 2007; Tosca et al. 2007; Downs 2011). The mechanism for the meiosis-inducing action of pyruvate in the absence of glucose has been enigmatic, because the logical explanation, namely that of transit through the tricarboxylic acid (TCA) cycle, does not appear to come into play. Intriguingly, this mechanism may also have ties to AMPK metabolism. NADH has been shown to suppress AMPK activity (Rafaeloff-Phail et al. 2004). When mouse DOs are maintained in meiotic arrest and stimulated with 50 -aminoimidazole-4carboxyamide-ribonucleoside (AICAR), an activator of AMPK, meiotic resumption is suppressed by NADH in dose-dependent manner (S. M. Downs, unpubl. obs.). In addition, COCs consume a large amount of pyruvate in culture, with a significant portion not targeted for oxidisation (Downs 1995). Rather, much of it is converted to lactate via the enzyme lactate dehydrogenase and, in so doing, NADH is oxidised to NADþ, which can replenish that lost during glycolysis and thereby keep that pathway active. However, it is possible that increased metabolism of pyruvate to lactate reduces NADH levels below the threshold required to block AMPK activation and the active kinase stimulates meiotic resumption. Confirmation of such a mechanism will require further testing. Definite differences exist in the regulation of rodent oocyte maturation. For example, when the effects of relative pyruvate and glucose levels were compared in 3-isobutyl-1-methylxanthine (IBMX)-arrested mouse and rat oocytes, opposite effects of these energy substrates were observed on meiotic maturation; indeed, high levels of pyruvate in the absence of glucose were inhibitory to rat oocyte maturation, instead of stimulatory, as was observed in the mouse (Downs 2011). This may be related

Energy and oocyte maturation

to the additional finding that stimulation of AMPK in rat oocytes has little positive action on meiotic resumption (Downs 2011). It should be noted that pronounced differences also exist in the metabolism of rat and mouse preimplantation embryos (e.g. Brison and Leese 1994). It is important to note that very high concentrations of glucose can also induce GVBD in arrested oocytes (Downs and Mastropolo 1994). The mechanism for this is not clear, but the excessive glucose may force a Crabtree effect, where glucose is diverted from the TCA cycle to lactate production or to other pathways not normally active. Increased lactate production would reduce NADH levels, possibly to the extent that AMPK is released from an inhibitory influence (see above), thereby negating the effect of glycolytic ATP production and leading to a non-physiological meiotic response. The culture medium we and numerous laboratories routinely use for oocyte culture is Eagle’s minimum essential medium (MEM), which contains salts, amino acids and vitamins. For energy, the medium is supplemented with glucose and pyruvate, but also contains another potential energy substrate: glutamine. This amino acid has been shown to support the spontaneous maturation of rabbit and hamster oocytes (Gwatkin and Haidri 1973; Bae and Foote 1975) and is more effective than pyruvate in augmenting glucose-supported maturation in bovine oocytes (Krisher and Bavister 1998). However, glutamine alone cannot support mouse oocyte maturation, because oocytes either arrest at prophase I or die when glutamine is the only energy source (Bae and Chung 1975; Fagbohun and Downs 1992). Glutamine nevertheless has a notable influence on maturation depending on the type of culture medium and the supplements therein. In MEM supplemented with meiotic inhibitor, glutamine has a strong inhibitory effect on CEOs in the absence of glucose but no additional effect in its presence. Conversely, in M16 medium (a simple buffered salt solution lacking the vitamin and amino acids present in MEM) glucose has no effect on meiotic arrest, whereas glutamine is profoundly inhibitory (Downs and Verhoeven 2003). These meiosis-suppressing actions of glutamine and glucose can be traced to their augmentation of de novo synthesis of inhibitory purines; in fact, phosphoribosylpyrophosphate, the product of glucose transit through the pentose phosphate pathway (PPP), is the starting substrate for purine de novo synthesis and purine salvage, and the amino acids glutamine, aspartate and glycine all contribute to the purine backbone. When present in culture medium, these molecules augment purine levels in mouse COCs (Downs 1998). Furthermore, these actions are manifest in CEOs, but not DOs, underscoring not only significant differences in metabolism between the two types of oocytes, but also the importance of the somatic compartment in meiotic regulation. The increased purine production presumably leads to production of purine nucleotides that feed into meiosis-arresting pathways (for reviews, see Downs 2010; Jaffe and Norris 2010). Thus, effective meiotic arrest depends on the coordinated interaction between two metabolic pathways (Fig. 2). In a recent publication, Wigglesworth et al. (2013) demonstrated yet another example of oocyte-directed metabolic cooperation between the mouse oocyte and its attendant cumulus

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cells. Secreted oocyte factors increase cumulus cell expression of inosine monophosphate (IMP) dehydrogenase, the first enzyme in the pathway to guanyl compounds from IMP, the end-product of de novo purine synthesis. The action of IMP dehydrogenase promotes the production of cGMP, a critically important cyclic nucleotide transmitted to the oocyte through gap junctions that helps maintain elevated cAMP levels within the oocyte, thereby preventing premature meiotic resumption. Thus, the oocyte indirectly controls its own meiotic destiny. Glucose metabolism and meiotic induction Many studies of oocyte maturation have been conducted using a spontaneous maturation system, but such conditions do not duplicate those occurring in vivo. Although spontaneous maturation in vitro occurs simply by removing the meiosis-inhibiting influence of the follicle, maturation in vivo is a more active process requiring a gonadotrophin-induced stimulus that overturns extant inhibitory signals. Moreover, the induction process requires participation of the granulosa cells, for they are indirect mediators of the gonadotrophin-directed meiotic response. Thus, the events underlying meiotic induction give a clearer physiological picture of what transpires during meiotic resumption in situ. The isolated CEO has been a valuable model system for the study of meiotic induction. To prevent spontaneous maturation, the membrana granulosa is replaced by meiotic inhibitors and maturation is stimulated by gonadotrophin, such as FSH (Downs et al. 1998), or the recently discovered downstream hormonal mediators epidermal growth factor (EGF)-like proteins (Downs et al. 1998; Park et al. 2004; Sekiguchi et al. 2004). Unfortunately, the use of this approach for oocytes from non-rodent species is less practical, for it is usually ineffective in maintaining and/or promoting good viability and developmental competence (Gilchrist and Thompson 2007). Using this model system in mice it has been shown that glucose is indispensable for meiotic induction (Fagbohun and Downs 1992; Downs and Mastropolo 1994). In response to hormone treatment, hexokinase activity in COCs is increased and glucose is converted to glucose-6-phosphate, no doubt contributing to the fourfold stimulation of uptake by complexes (Downs et al. 1996). Further metabolism is crucial, because meiotic resumption is prevented by 2-deoxyglucose (2-DG), a glucose analogue that is metabolically inert following its phosphorylation (Downs et al. 1996). Such metabolism can take one of several routes: through the glycolytic, pentose phosphate, hexosamine or sorbitol pathways (Sutton-McDowall et al. 2010; Fig. 2). Although glycolysis is stimulated in COCs by gonadotrophin (Zuelke and Brackett 1992; Downs and Utecht 1999) and EGFlike peptides (Richani et al. 2014), several lines of evidence indicate that this pathway does not mediate the meiotic response in oocytes: (1) blocking glycolysis with 2-DG or iodoacetate fails to block hormone-induced maturation (Tsafriri et al. 1976; Downs et al. 1996); (2) pyruvate alone is unable to stimulate ATP synthesis in the presence or absence of FSH (Downs 1995); and (3) neither high pyruvate levels nor hormonal stimulation increase oxygen consumption (Dekel et al. 1976; Magnusson

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cGMP Polyol

Glucose HK

Fructose

GTP

NADPH NADP NAD NADH

PPP

Glucose-6-P

PRPP

NADP NADPH NADP NADPH

IMP

sis oly

cAMP

am ine

Gl yc

os

Lactate

ATP

x He

NAD NADH

PDE3

O-linked glycosylation

Pyruvate

Acetyl CoA

HX

De Novo Purine

Fructose-6-P AMPK∗?

PRPP

Glutamine AICAR

5⬘-AMP

Hyaluronic acid AMPK∗

AMPK

Fatty Acyl CoA FAO

TCA cycle

AICAR

Acetyl CoA Mitochondrion

Fig. 2. Interconnected pathways of glucose metabolism. Glucose phosphorylation by hexokinase (HK) generates glucose-6-P, which can be metabolised to fructose-6-P followed by transit through either glycolysis or the hexosamine pathway, which leads to O-linked glycosylation or hyaluronic acid synthesis. Glycolysis produces pyruvate, which can be oxidised through the tricarboxylic acid (TCA) cycle or converted to lactate. It is possible that the latter pathway could help generate active AMP-activated protein kinase (AMPK) via oxidation of NADH. An alternative pathway for glucose-6-P is the pentose phosphate pathway (PPP), which produces NADPH and phosphoribosyl pyrophosphate (PRPP), the starting molecule for de novo purine synthesis. Coordinated activity of these two pathways is thought to generate purines, which can have either inhibitory or stimulatory actions on oocyte maturation. Inosine monophosphate (IMP), the product of de novo purine synthesis, can be converted to both guanyl and adenyl compounds, notably cGMP and cAMP, which are important in signalling pathways regulating meiotic maturation. Degradation of cAMP in the oocyte by phosphodiesterase (PDE) 3 produces 50 -AMP, a potent activator of AMPK, a kinase that, when active (*), triggers germinal vesicle breakdown in mouse oocytes by stimulating fatty acid oxidation (FAO) in the mitochondria. Excessive glucose levels can result in hexose bypassing hexokinase and entering the polyol pathway to produce fructose. HX, hypoxanthine; AICAR, 50 -aminoimidazole-4-carboxyamide-ribonucleoside.

et al. 1977; Downs et al. 1997). Thus, increased respiration driven by glycolytically produced pyruvate is not the driving force behind meiotic induction, but increased oxygen consumption following GVBD indicates its contribution to the completion of meiosis (Johnson et al. 2007). It is important to note that lower concentrations of 2-DG that block glycolysis (1 mM) do not prevent meiotic induction, but higher concentrations ($5 mM) do and are presumably necessary to prevent PPP activity (see below). One example of a disconnect between nuclear and cytoplasmic maturation is that although gonadotrophin, EGF and EGF-like peptides are equally effective in meiotic induction, EGF-like peptides more effectively promote developmental potential in mouse CEOs (Richani et al. 2013). Monitoring glucose metabolism during hormone-stimulated mouse oocyte maturation indicated that the PPP is a major participant in meiotic resumption. Experiments with radiolabelled glucose showed that FSH triggered increased oxidation

of glucose by its transit through the PPP and not via glycolysis (Downs and Utecht 1999). Moreover, pharmacological modification of the PPP demonstrated that its activity was associated with meiotic resumption (Downs et al. 1998). A prominent role for this pathway in nuclear maturation is supported by additional studies in mice and other species (Urner and Sakkas 1999, 2005; Herrick at al 2006; Alvarez et al. 2013; Gutnisky et al. 2014). Kim et al. (2012) reported that the PPP is not involved in GVBD in mouse oocytes but, rather, progression beyond MI; however, they tested spontaneous maturation in DOs as opposed to meiotic induction in CEOs, as was the case in our studies. Interestingly, PPP activity in the rat oocyte appears to diminish during meiotic maturation (Tsutsumi et al. 1992). One mole of glucose metabolised through the oxidative arm of the PPP generates two moles of NADPH and one mole of ribose-5-P. NADPH contributes significantly to the reduction equivalents in the oocyte, being used in most anabolic reactions

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Table 2. Effect of culture media on meiotic maturation of mouse oocytes Data are from Downs and Mastropolo (1997). NA, not attempted; GVBD, germinal vesicle breakdown; db-cAMP, dibutyryl-cAMP; HX, hypoxanthine; MEM, minimum essential medium Culture medium

MEM F-10 M199 M16 MB752/1 L-15

Spontaneous maturation

Meiotic arrest

Meiotic induction

GVBD

MII

db-cAMP

HX

db-cAMP

HX

þþþ þþþ þþþ þþþ þþþ þþþ

þþ þþ þþ þþ þ þþþ

þþþ þþþ þþþ þ þþþ þþþ

þþ þ þþ No arrest No arrest þþþ

þþþ þþþ þþþ þ þþ No induction

þþþ þþþ þþþ NA NA þþ

and serving as an important antioxidant. NADPH is used to regenerate glutathione (an important protectant against reactive oxygen species) and accumulates during cytoplasmic maturation, but the source of this NADPH in mouse oocytes does not appear to be the PPP (Dumollard et al. 2007). Ribose-5-P is important in the production of nucleotides and nucleic acids, and it is the production of phosphoribosyl pyrophosphate (PRPP) in the non-oxidative arm of the PPP and its participation in de novo purine synthesis that contributes to meiotic induction. Indeed, treating hormone-stimulated mouse CEOs with inhibitors of purine synthesis blocks meiotic induction, an effect reversed by treatment with compounds such as AICAR that enter into the purine synthesis pathway downstream of the site of inhibition (Downs 1997a, 1997b). These findings appear contradictory to the studies cited earlier that suggested a role for purine synthesis in the maintenance of meiotic arrest. However, these opposite effects can be explained when one considers the timing and duration of the purine synthetic response. Whereas meiotic arrest necessitates the chronic presence of an inhibitory level of cAMP within the oocyte, aided by a steady level of glucose metabolism through the PPP, meiotic induction occurs as a result of a gonadotrophindriven transient pulse of metabolic activity, producing levels of nucleotide well above this inhibitory threshold and initiating a signal cascade that drives oocyte cAMP below the inhibitory threshold.

db-cAMP or HX, no doubt due to its lack of amino acids. HX failed to maintain meiotic arrest in Waymouth’s MB752/1 medium due, at least in part, to the lack of pyruvate. Finally, in Leibovitz’s L-15 medium, FSH and EGF were able to stimulate meiotic resumption in HX-arrested CEOs, but FSH was unable to induce maturation in db-cAMP-arrested oocytes. This may be due to differing abilities of CEOs to synthesize purines. L-15 medium is unusual in that glucose is replaced with galactose and the pyruvate concentration is very high (5.1 mM in L-15 medium vs 0.23 mM in MEM). We hypothesised that this unusual energy substrate content could explain the deficit in meiotic induction in db-cAMP-supplemented medium. When the galactose in L-15 medium was replaced with 5.5 mM glucose, no increase in meiotic resumption was observed in response to FSH; the same result was observed when the pyruvate concentration was reduced to 0.23 mM. However, when both changes were made, a significant 40% increase in maturation was realised (Fig. 3). It is tempting to speculate that the lack of glucose limited purine synthesis, whereas the presence of high pyruvate suppressed glucose metabolism. In addition, enough PRPP may have been present to support nucleotide formation via HX salvage but not de novo synthesis, thus explaining the ability of FSH to stimulate GVBD in HXsupplemented medium. These observations serve as a powerful example of the influence culture media and the proper balance of energy substrates have on oocyte meiotic regulation.

Comparison of culture medium effects on meiotic maturation

A new focus in meiotic regulation

As already discussed, the choice of culture medium plays a crucial role in the analysis of meiotic regulation and is a prime consideration when devising experiments on oocyte maturation. To examine this idea more comprehensively, we tested six different culture media on three parameters of oocyte maturation: (1) meiotic progression to MII during spontaneous maturation; (2) maintenance of meiotic arrest in the presence of dibutyryl-cAMP (db-cAMP) or hypoxanthine (HX); and (3) meiotic induction by FSH or EGF (data from Downs and Mastropolo 1997). These results are summarised in Table 2. MEM, F-10, and M199, all complex media, comparably supported spontaneous maturation, meiotic arrest and meiotic induction. M16 was a poor medium for maintaining meiotic arrest by either

Oocytes from domestic species differ from rodent oocytes in that they contain significantly more lipid (Loewenstein and Cohen 1964; McEvoy et al. 2000). Consequently, there has been increased attention focused on lipid metabolism as it relates to oocyte development in these non-rodent species (for reviews, see Sturmey et al. 2009; Dunning et al. 2014). Lipids, stored as triglycerides, are a ready source of energy (Homa et al. 1986) and are oxidised by bovine and porcine oocytes (Ferguson and Leese 1999; Kim et al. 2001; Sturmey and Leese 2003). Studies have shown a dependence of oocytes on FAO, because blocking FAO in maturing oocytes with methyl palmoxirate or mercaptoacetate compromises subsequent development (Sturmey and Leese 2003; Ferguson and Leese 2006; Sturmey et al. 2006). Bovine oocytes can complete maturation in vitro in the absence

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⫺FSH

S. M. Downs

Meiotic stimulus

⫹FSH

80

(AICAR), 5⬘AMP

cAMP

Percent GVBD

70 AMPK∗

AMPK

60 50

ACAC

40

Long chain fatty acids

Malonyl CoA

Acetyl CoA

30 20

Fatty Acyl CoA Carnitine

10

CoA

0 MEM

Gal 5.5 Pyr

Gal 0.23 Pyr

Glc 5.5 Pyr

Glc 0.23 Pyr

C75 Mercaptoacetate Etomoxir

CPT1 CoA Carnitine

Fatty acylcarnitine CPT2

Fatty acyl CoA

L-15

Betaoxid

Acetyl CoA

Mitochondrion

Fig. 3. Effects of energy substrate composition in L-15 medium on FSHinduced maturation. Mouse cumulus cell-enclosed oocytes (CEOs) were cultured for 17–18 h in dibutyryl-cAMP (db-cAMP)-supplemented minimum essential medium (MEM) or Leibovitz’s L-15 medium to maintain meiotic arrest and treated with FSH to stimulate maturation. FSH stimulated most of the arrested oocytes to undergo germinal vesicle breakdown (GVBD), whereas it had no effect in L-15 medium (galactose þ 5.5 mM pyruvate). When either galactose was exchanged for 5.5 mM glucose or pyruvate was reduced to 0.23 mM, FSH failed to stimulate maturation, but when both changes were made FSH stimulated significant meiotic resumption. Data adapted from Downs and Mastropolo (1997); this material is reproduced with permission of John Wiley and Sons, Inc.

Fig. 4. Regulation of fatty acid oxidation by AMP-activated protein kinase (AMPK). When gonadotrophin stimulates the resumption of maturation, it promotes the degradation of oocyte cAMP to 50 -AMP, a potent stimulator of AMP, a stimulator of AMPK (*). 50 -Aminoimidazole-4-carboxyamideribonucleoside (AICAR) is another activator of AMPK. AMPK phosphorylates, and thereby inactivates, acetyl CoA carboxylase (ACAC), leading to reduced levels of malonyl CoA. This removes a block to carnitine palmitoyltransferase (CPT) 1, which adds a carnitine residue to long-chain fatty acids for entry into mitochondria. Subsequent exchange of carnitine for CoA by CPT2 converts the fatty acid to fatty acyl CoA, which is then oxidised. Figure adapted from Valsangkar and Downs (2013).

of exogenous energy substrates, suggesting that they draws on endogenous lipid reserves to do so (Ferguson and Leese 2006), an adaptation not available to the mouse oocyte (Downs and Hudson 2000). That such an energy source fuels meiosis is supported by the findings that oocyte lipid levels decrease during the course of maturation (Ferguson and Leese 1999; Kim et al. 2001; Sturmey and Leese 2003) but remain elevated when FAO and maturation are blocked (Paczkowski et al. 2013). A role for lipid in mouse oocyte physiology is not intuitive due to the paucity of lipid present in this species. However, it is important to note that a small amount of triglyceride, comprised of three fatty acid chains linked to a glycerol backbone, can provide a tremendous amount of energy; where one glucose molecule yields 27–31 molecules of ATP, 104 molecules of ATP are produced from one molecule of the fatty acid palmitate (cf. Sturmey et al. 2009). We were led to investigate the potential role of FAO in mouse oocyte maturation because of our work with AMPK. An important substrate of AMPK is acetyl CoA carboxylase (ACAC; Davies et al. 1992), a ratelimiting enzyme in fatty acid synthesis that produces malonyl CoA from acetyl CoA. AMPK phosphorylation inactivates ACAC and lowers malonyl CoA levels (Tong 2005); the loss of malonyl CoA removes its inhibition of carnitine palmitoyltransferase 1 (CPT1), the enzyme that catalyses the replacement of acetyl CoA on large chain fatty acids with carnitine, in turn promoting their entry into mitochondria and turning on FAO (Ruderman and Prentki 2004; see Fig. 4). Consequently, AMPK is a potent activator of FAO.

A study incorporating pharmacological modifiers of FAO showed that treating meiotically arrested oocytes with activators of this pathway (including AICAR, an activator of AMPK, or C75, an activator of CPT1) triggered GVBD, whereas blocking this pathway with the CPT1 inhibitors malonyl CoA or etomoxir prevented meiotic induction (Downs et al. 2009). These surprising results established that FAO was required for meiotic induction by pharmacological means; further experiments confirmed a similar relationship for FSH- and EGF-induced maturation (Valsangkar and Downs 2013). A study wherein the effects of etomoxir on bovine, porcine and murine oocytes were compared revealed that etomoxir blocked maturation in all three species, but the concentration required was indirectly related to lipid content (Paczkowski et al. 2013). This indicates that the oocytes have a varied reliance on lipids as energy sources; indeed, porcine oocytes, which contain the greatest amount of lipid among the three, are least able to adapt to etomoxir inhibition with glycolysis-derived energy (Paczkowski et al. 2013; see also Ferguson and Leese 2006; Sturmey and Leese 2008). The impact of lipid metabolism in rodent oocyte development has recently become an active area of research. Kylie Dunning and colleagues have performed an important series of studies examining the role of lipids in mouse oocyte development. They report that b-oxidation is an essential component of mouse oocyte developmental competence and that blocking FAO during oocyte maturation has serious consequences for

Energy and oocyte maturation

subsequent embryo development (Dunning et al. 2010), similar to the effects reported in domestic species. FAO occurs in both oocytes and cumulus cells, but at much higher levels in cumulus cells, once again highlighting the importance of the somatic compartment in providing metabolites for oocyte consumption. In addition, augmenting lipid metabolism by supplementing culture medium with agents such as L-carnitine, a molecule critical for rate-limiting fatty acid entry into mitochondria (Fig. 4), improved oocyte quality and increased embryo developmental rates (Dunning et al. 2011). Consistent with this, we have observed augmentation of meiotic induction by the addition of carnitine or palmitate to oocyte maturation medium (Downs et al. 2009; Valsangkar and Downs 2013). Because of the importance of lipid metabolism in establishing developmental competence, L-carnitine supplementation has become a popular modification to improve development of in vitromatured oocytes. A further indication of its importance in human IVF is that despite having the metabolic machinery for FAO, oocytes are unable to synthesis their own carnitine (Montjean et al. 2012; Me´ne´zo et al. 2013). Although there is little evidence that dietary carnitine is beneficial, IVF outcomes have consistently benefited from its inclusion in oocyte and embryo culture media, by way of increased b-oxidation and relief of oxygen stress (Dunning and Robker 2012). Such supplementation overcomes a deficiency of in vitro maturation by providing a beneficial nutrient available in the follicular environment (cf. Sutton et al. 2003). The significant contribution of FAO to oocyte maturation raises the question of the relative importance of carbohydrate and lipid energy-generating systems. A possible explanation has recently been offered by Krisher and Prather (2012) for embryo development that may also apply to oocytes. Krisher and Prather (2012) propose that a Warburg effect is invoked in which energy is provided by FAO while glucose metabolism is diverted away from energy generation into other pathways producing molecules important for cell division and defence against oxygen stress. They further propose that this scenario is compatible with the quiet embryo hypothesis, originally proposed by Henry Leese (2002), which states lower metabolic rates, accompanied by high antioxidant potential and active AMPK, are optimal for embryo viability. Such a relationship reflects the need to respond to culture stress and the importance of metabolic plasticity in oocytes and embryos. A future challenge will be to gain a better understanding of how different metabolic pathways interact to enable our efficient manipulation of metabolism in achieving optimal developmental success. References Alvarez, G. M., Ferretti, E. L., Gutnisky, C., Dalvit, G. C., and Cetica, P. D. (2013). Modulation of lycolysis and the pentose phosphate pathway influences porcine oocyte in vitro maturation. Reprod. Domest. Anim. 48, 545–553. doi:10.1111/RDA.12123 Bae, I.-H., and Chung, S.-O. (1975). The in vitro maturation of the mouse oocyte. Yonsei Med. J. 16, 18–28. doi:10.3349/YMJ.1975.16.1.18 Bae, I.-H., and Foote, R. H. (1975). Carbohydrate and amino acid requirement and ammonia production of rabbit follicular oocytes matured in vitro. Exp. Cell Res. 91, 113–118. doi:10.1016/0014-4827(75) 90148-2

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