Reprod Dom Anim 45 (Suppl. 3), 32–41 (2010); doi: 10.1111/j.1439-0531.2010.01662.x ISSN 0936-6768

Nutritional and Metabolic Mechanisms in the Ovary and Their Role in Mediating the Effects of Diet on Folliculogenesis: A Perspective RJ Scaramuzzi1,2, HM Brown1 and J Dupont1 1 UMR Physiologie de la Reproduction et des Comportements, L’Institut National de la Reproduction, Nouzilly, France; 2Department of Veterinary Basic Sciences, Royal Veterinary College, Hertfordshire, UK

Contents Folliculogenesis in ruminants is a nutritionally sensitive process, and short-term increases in nutrient flux can stimulate folliculogenesis in sheep and cattle. These short-term effects are probably mediated directly at the follicular level to modify gonadotrophin-induced follicle growth and development. The follicle appears to have a number of ‘nutrient sensing’ mechanism that may form the link between nutrient status and folliculogenesis. This review examines the evidence for the presence of pathways that may sense nutrient flux from within the follicle including the insulin signalling pathway, adenosine monophosphate-activated kinase (AMPK), the hexosamine pathway, peroxisome proliferator-activated receptors (PPARs) and leptin. The review then assesses the available evidence concerning their mechanisms in the follicle and speculates on how these ‘nutrient sensing’ pathways are integrated into the FSH signalling pathways to adjust gonadotrophin-stimulated follicular function. We conclude that there is good evidence to suggest that the follicle does contain more than one functional ‘nutrient sensing’ pathway that have intra-follicular effects on some FSH-mediated functions such as the synthesis of oestradiol, in granulosa cells. These pathways include insulin, AMPK, and leptin. There is also a good case for the integration of PPARs in the intra-follicular sensing of nutrient flux. However, there is little evidence at present to suggest the hexosamine biosynthetic pathway has functional significance in the follicle as a sensor of nutrient flux. Further study will be required to fully understand ‘nutrient sensing’ pathways in the follicle and their cross-talk with FSH signalling pathways.

Introduction It is widely accepted that environmental influences on reproduction are integrated into homeostatic mechanisms that control reproduction, primarily at a hypothalamic level. Thus, the effect of photoperiod on the pattern of seasonal reproductive activity is controlled by the action of melatonin on the hypothalamus similarly the effect of severe negative energy balance on the secretion of GnRH by the hypothalamus. In the ewe, short-term nutritional supplementation stimulates folliculogenesis (Vin˜oles et al. 2005; Somchit et al. 2007) and increases ovulation rate (Teleni et al. 1989; Letelier et al. 2008), and it has been suggested that the mechanism of this effect is not primarily hypothalamic but that it involves direct effects of nutrients and metabolites on the follicle (Scaramuzzi et al. 2006). These data suggest that environmental effects, at least with respect to nutrition, can be integrated into follicular control mechanisms at an ovarian level further suggesting that there are specific metabolic sensing mechanisms in the follicle that can mediate nutritional influences on the follicle. This review will describe known metabolic

mechanisms in the follicle, and it will consider how intra-ovarian metabolic mechanisms can be integrated into known reproductive control systems. Folliculogenesis is a nutritionally responsive process that adapts to direct and indirect nutritional signals. Increased nutrition stimulates folliculogenesis, and there is now strong evidence to show that follicles respond to direct actions of nutrition, and there is little evidence to show that nutrition stimulates folliculogenesis indirectly by increasing the secretion of gonadotrophins. Conversely, under-nutrition inhibits folliculogenesis, and in this case, there is compelling evidence to show that undernutrition reduces LH and FSH secretion by central actions on the hypothalamo-pituitary system and particularly by the inhibition of the GnRH pulse generator. These differences suggest that the effects of undernutrition and over-nutrition on folliculogenesis are not the opposite extremes of a single mechanism; rather they are two different mechanisms. This has important implications for both the design and interpretation of experiments to examine the effects of nutrition on fertility.

Nutritional Influences on the Follicle The main component of diet that stimulates folliculogenesis is energy, in particular glucose, although energy derived from fatty acid oxidation also appears to be important. Proteins, vitamins and other micronutrients probably exert permissive rather than regulatory functions on folliculogenesis. Consequently, research interest has concentrated on associations and functional links between the regulation of carbohydrate and fatty acid metabolism and the regulation of folliculogenesis. The physiological link between energy intake and folliculogenesis most probably involves several metabolic hormones and growth factors including insulin, IGF-I, leptin and growth hormone (GH) acting within the follicular environment to modulate gonadotrophinstimulated folliculogenesis. In this model, the central role of gonadotrophins in regulating folliculogenesis is recognized, and the model suggests that the metabolic regulators of carbohydrate metabolism and fatty acid oxidation have signalling functions that locally modify gonadotrophic actions in the follicle.

Intra-follicular Metabolic Mechanisms The effects of nutritional supplementation on folliculogenesis and ovulation rate have been widely investigated in farm animals where there is a strong case for the use of targeted nutritional supplementation as a non 2010 Blackwell Verlag GmbH

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hormonal means of increasing prolificacy and fertility of farm animals (Scaramuzzi and Martin 2008). Recent research has focussed on the intra-follicular responses to nutritional and metabolic stimuli and has shown that the follicle can directly sense nutritional and metabolic inputs and modify its responses accordingly. Simply, the follicle has specific ‘nutrient sensing’ and metabolic mechanisms that can modify folliculogenesis. ‘Nutrient sensing’ is a term that has come into vogue in recent times, and ‘nutrient sensing’ mechanisms have been described in muscle and adipose tissue. Much of this research has been on non-reproductive tissues particularly in relation to the pathophysiology of Type II diabetes mellitus, and several ‘nutrient sensing’ pathways have been identified, and their mechanisms partly defined. Pathways and molecules that have been implicated in ‘nutrient sensing’ include adenosine monophosphate kinase (AMPK) often referred to as the ‘master metabolic switch’, the mammalian target of rapamycin (mTOR), malonyl CoA and the hexosamine pathway (Marshall et al. 1991; Marshall 2006). Obvious questions arise concerning the functionality of these pathways in the follicle and their role in mediating direct nutritional effects on folliculogenesis, and these will be examined with respect to folliculogenesis in the following sections. Glucose Glucose is an essential fuel for metabolism, and its status is sensed locally by a number of intra-cellular mechanisms that are probably inter-related. These include insulin-signalling, the actions of AMPK and the hexosamine pathway. The blood concentrations of glucose in ovarian venous blood are lower than in carotid arterial blood (Scaramuzzi et al. 2010), indicating that the ovary actively takes up glucose from the circulation. The glucose transport proteins, GLUT1 and GLUT4, have also been reported in granulosa and theca cells from sheep and cattle (Williams et al. 2001; Nishimoto et al. 2006). Admittedly, the presence of GLUTs in the follicle is not unexpected given that, like other tissues, the follicle needs glucose to generate ATP. What is perhaps slightly surprising is the presence of GLUT4 in the follicle; GLUT-4 is an insulin-dependent transporter with a low affinity, but a high capacity, for glucose transport in those tissues with a high demand for glucose (typically, skeletal and cardiac muscle) or adipose tissue where excess glucose is stored as triacylglyceride. The presence of GLUT4 in the follicle suggests that the roles of insulin and of the insulinmediated uptake of glucose in the follicle are atypical. Insulin signalling in the follicle The follicle has a functional insulin–glucose system (Fig. 1). Insulin receptors have been identified in the follicle (Poretsky et al. 1999) with insulin signalling primarily through the insulin receptor rather than the closely related IGF-I receptor (Willis and Franks 1995). The insulin receptor is a receptor tyrosine kinase that autophosphorylates on insulin binding. The effects of insulin on cellular function are mediated by an  2010 Blackwell Verlag GmbH

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interconnecting array of intracellular pathways (Taniguchi et al. 2006) involving several phosphatases and intermediary and terminal kinases as well as numerous scaffold and docking proteins that impart hormone and tissue specificity on the cellular responses to insulin. The insulin receptor substrate proteins (IRS-1, IRS-2 and IRS-4) interact with the insulin receptor and when phosphorylated by the ligand-activated receptor initiate insulin’s intracellular responses. The IRSs have been detected in human granulosa cells (Yen et al. 2004) and in both granulosa and theca cells in the ewe (Somchit 2008). In women, a mutation of the IRS-2 gene has been linked to polycystic ovarian disease (Ehrmann et al. 2002). The follicle expresses other kinases activated by insulin, although not exclusively, including PI3K, Akt, P70S6K, mTOR and the ERKs (Zeleznik et al. 2003; Ryan et al. 2007, 2008). The phosphatase, PTEN, has been identified in ovine follicles (Froment et al. 2005). In mice whose oocytes lack PTEN, the entire pool of primordial follicles is activated. Consequently, primordial follicles are depleted, resulting in premature ovarian failure (Reddy et al. 2008). The target of rapamycin (mTOR) ⁄ p70 S6 kinase (S6K) pathway is probably a ‘nutrient sensing’ mechanism in the follicle. The kinase, mTOR, is expressed in the mouse ovary, and its inhibition reduced granulosa cell proliferation and follicle growth (Yaba et al. 2008). The tumour suppressor tuberous sclerosis complex 2 (Tsc2) negatively regulates mTOR and maintains the dormancy of primordial follicles. Primordial follicles are activated in mutant mice lacking the Tsc2 gene in their oocytes as a result of elevated mTOR, thus causing premature ovarian failure (Adhikari et al. 2008). Overall, there is no doubt that the follicle contains a functional insulin–glucose system, but what is the role of insulin in folliculogenesis? There are at least two possibilities. The first and simplest is that insulin has essential non-specific functions in the maintenance of cellular health and integrity in follicular cells as it does in all other cells. Second, the insulin–glucose system may also have effects specific to granulosa and theca cells. This later possibility is supported by a large body of evidence, derived principally from in vitro studies showing that insulin has specific actions during folliculogenesis, perhaps even direct gonadotrophic effects (Poretsky and Kalin 1987). The separate actions of glucose and insulin because of their physiological interdependence are difficult to unravel. In vitro studies utilizing serum-free systems for culturing granulosa cells (Campbell et al. 1996) and theca cells (Campbell et al. 1998) have demonstrated beyond doubt that basal and gonadotrophin-stimulated cellular proliferation and steroidogenesis are increased in a dose-dependent manner by insulin (Campbell et al. 1996). There is also broad agreement among these studies and others using alternative in vitro models such as granulosa cells cultured in undefined media or the culture of granulosa cells derived from post-LH surge follicles. However, in vitro studies using granulosa cells generally investigate the role of insulin in an excess of glucose. The effects of insulin in vivo are not entirely consistent with in vitro findings particularly in relation to follicular

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Fig. 1. A summary (using standardized nomenclature) of insulin signalling pathways in the follicle. Insulin can signal through several interconnecting pathways to affect a wide variety of functions in the granulosa cell including, cell survival and proliferation, protein synthesis and cell differentiation as well GLUT4 (glucose transporter 4) mediated uptake of glucose. The main pathways illustrated are the ERK (extra cellular related kinase), mammalian target of rapamycin (mTOR); PI3K (phosphatidylinositol-3 kinase) and Akt (a member of the serine ⁄ threonine-specific protein kinase family) pathways. Arrows indicate stimulatory pathways, ‘T’ endings indicate inhibitory pathways, and dashed lines indicate terminal events in a pathway

steroidogenesis. Published data from in vivo experiments do not provide convincing evidence that insulin stimulates follicular steroidogenesis in situ (Poretsky et al. 1999). Data from in vivo studies with animal models have shown that insulin, or treatments that acutely increased insulin, increased the number of follicles and at the same time, reduced the expression of Aromatase in granulosa cells of oestrogenic follicles (Somchit 2008; Gallet et al. 2009) and the circulating concentrations of oestradiol (Peluso et al. 1991; Letelier et al. 2008; Gallet et al. 2009). These observations raise a legitimate unanswered question over the contribution of in vitro studies to our understanding of the function of insulin in follicular physiology in situ, that remain to be resolved. Adenosine monophosphate-activated kinase (AMPK) Adenosine monophosphate-activated kinase is a serine ⁄ threonine kinase that is activated by an increase in the ratio of AMP to ATP which is associated with a depletion of ATP in response to nutritional and environmental stress (Hardie 2004). It is made up of three subunits, a catalytic a sub-unit that has two isoforms (a1 and a2) and constitutive b and c sub-units that have either two (b1 and b2) or three (c1, c2 and c3) isoforms. In peripheral tissues, AMPK is involved in several intracellular, metabolic pathways including cellular uptake of glucose, glycolysis, the oxidation of fatty acids, and the synthesis of sterols (Carling et al. 1987). Adenosine monophosphate-activated kinase is also implicated in ovarian function in several species; the various subunits have been identified in granulosa cells, theca cells and oocytes (Tosca et al. 2008). In bovine granulosa cells in vitro, two activators of AMPK, 5aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) and N, N-dimethylimidodicarbonimidic diamide (metformin) inhibit the secretion of both proges-

terone and oestradiol (Tosca et al. 2008), and in granulosa cells isolated from immature rat ovaries FSH-induced cell proliferation was inhibited by AMPK (Kayampilly and Menon 2009). These effects are associated with the inhibition of the mitogen-activated protein kinase and extracellular-regulated kinase (MAPK; ERK1 ⁄ 2) signalling pathway. Adenosine monophosphate-activated kinase is often viewed as a ‘metabolic master molecule,’ and in the ovary it may regulate metabolic influences on folliculogenesis and oocyte maturation (Fig. 2). Indeed, many of the metabolic hormones involved in the control of reproductive function such as glucose and leptin and the lesser studied resistin, adiponectin, and ghrelin can all act through the AMPK signalling cascade (Mitchell et al. 2005). Thus, the AMPK signalling pathways is a potential modulator of interactions between energy balance and folliculogenesis (Fig. 2). The hexosamine pathway The hexosamine biosynthetic pathway has been proposed as a cellular ‘sensor’ of energy status (Marshall et al. 1991) that can mediate the effects of glucose on the expression of several genes. The hexosamine ‘nutrient sensing’ system has been reported in muscle and adipose tissue (McClain et al. 1992; Wang et al. 1998). Following uptake and phosphorylation to glucose-6-phosphate, glucose is utilized along two major pathways: glycogen synthesis and the generation of ATP via glycolysis or the TCA cycle. However, a small amount of glucose is converted to fructose-6-phosphate and enters the hexosamine biosynthetic pathway (Fig. 3). The first step in this pathway is the conversion of fructose-6-phosphate to glucosamine-6-phosphate by the enzyme glutamine:fructose-6-phosphate amidotransferase. The end-product of the hexosamine pathway UDP-N-acetyl glucosamine (UDP-Glc-NAc) is the  2010 Blackwell Verlag GmbH

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Fig. 2. The mechanisms by which Metformin (N, N-dimethylimidodicarbonimidic diamide) activation of adenosine monophosphate activated kinase reduced steroidogenesis in bovine granulosa cells by inhibiting the mitogen-activated protein kinase (ERK) pathway leading to reduced concentrations of steroid acute regulatory protein (StAR) and the steroidogenic enzymes, 3bHSD and P450SCC and thus the secretion of ovarian steroid hormones

principal substrate for both O-linked and N-linked glycosylation of proteins. The flux of glucose in the hexosamine biosynthetic pathway regulates the expression of several genes whose products have effects of follicular function. For example, the infusion of either glucose or glucosamine increased the gene expression of TGFß and FGF2 in aortic smooth muscle cells (McClain et al. 1992) and TGFa in skeletal muscle (Daniels et al. 1993). The

Fig. 3. The hexosamine biosynthetic pathway and other pathways of glucose metabolism in the follicle. Most glucose is utilized for energy generation via intermediary metabolism; however, a small amount is quantitatively diverted via fructose-6-P, to glucosamine-6 phosphate from where it enters the hexosamine biosynthetic pathway where it can act as a nutrient sensor to alter gene transcription and glycosylation states

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follicular actions of TGFß, FGF2 and TGFa and can be summarized as follows. Both TGFa and FGF2 will inhibit Aromatase in vitro (Harlow et al. 1995) and the secretion of oestradiol 17ß in vivo (Campbell et al. 1994; Scaramuzzi and Downing 1995). The effects of glucosamine, an intermediate product of he hexosamine biosynthetic pathway, on gene transcription are mediated by O-linked glycosylation of the transcription factor Sp1, glycosylation serving to protect Sp1 from proteolytic degradation (Han and Kudlow 1997). The presence of Sp1 in the ovary has been confirmed (Borroni et al. 1997). In a recent study (Mun˜oz-Gutie´rrez 2005), we have shown that glucosamine infused into sheep at 10 mM ⁄ hz for 3 days increased the number of large follicles suggesting that this ‘nutrient sensing’ pathway may mediate nutritionally stimulated folliculogenesis. Hexosamine biosynthesis is active in bovine follicles and is particularly important for the production of hyaluronic acid during cumulus expansion, and perturbations in glucose flux through this pathway can reduce oocyte competence (Thompson et al. 2007). These observations suggest that this quantitatively minor pathway of glucose utilization has the potential to ‘sense’ metabolic status in the follicle, thus allowing the follicle to adapt to short-term variations in nutritional flux. Leptin The components of a functional leptin system have been observed in the follicle. In ruminants, leptin and its mRNA have been detected in theca cells and the oocyte (Ryan et al. 2002; Mun˜oz-Gutie´rrez et al. 2005; Pisani et al. 2008). In granulosa cells, leptin was only weakly expressed, and attempts to identify its mRNA have not been successful (Mun˜oz-Gutie´rrez et al. 2005; Pisani et al. 2008). The functional long form of the leptin receptor and its mRNA have also been detected in granulosa cells, theca cells and the oocyte (Mun˜ozGutie´rrez et al. 2005; Pisani et al. 2008). These findings suggest that leptin in addition to its endocrine functions

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may also have paracrine and possibly autocrine functions in the follicle. The predominant in vivo model examining the effects of leptin on reproduction has been the mutant obese (Ob ⁄ Ob) mouse. There have also been a very large number of descriptive studies, measuring leptin in blood and follicular fluid or describing the presence and distribution of leptin, its mRNA and the various forms of its receptor in reproductive tissues. The few available physiological studies suggest that exogenous leptin stimulates folliculogenesis in sheep (Kendall et al. 2004; Mun˜oz-Gutie´rrez et al. 2005). The effect of leptin on folliculogenesis and leptin-stimulated steroidogenesis has been examined in the ewe. Passive immunization of ewes against leptin increased the secretion of oestradiol and, conversely, infusion of leptin directly into the ovarian artery reduced ovarian oestradiol secretion (Kendall et al. 2004). The intrafollicular actions of leptin have been studied in vitro using cultured granulosa cells, and the published data suggest that leptin inhibits hormonally stimulated oestradiol production by granulosa cells in vitro (Zachow and Magoffin 1997) and by the follicle in vivo (Kendall et al. 2004). The inhibitory influence of leptin on oestradiol secretion appears to involve the IGF system; immuno-neutralization of IGF-I reverses the inhibitory effect of leptin on oestradiol secretion (Sirotkin et al. 2005), and leptin inhibits the effect of IGF-I on FSH-stimulated oestradiol production by granulosa cells (Zachow and Magoffin 1997; Spicer et al. 2000). Similarly, it inhibits the effect of IGF-I on LH-stimulated androstenedione production by theca cells (Spicer et al. 2000). Growth hormone and IGF-I The list of metabolic hormones that have direct effects on follicular function also includes GH. While the blood concentrations of leptin, glucose and insulin are all elevated when animals are in positive energy balance, GH is not, and in fact it is elevated during negative energy balance (Downing et al. 1995) when its principal function is to mobilize the bodies’ energy reserves to combat the effects of negative energy balance. Thus, when in negative energy balance, GH could potentially convey negative metabolic information to the follicle because the follicle contains GH receptors (Eckery et al. 1997). The signalling role of GH is complicated by two other facts, first GH stimulates the production of hepatic IGF-I and second prolonged exposure to high concentrations of GH can induce insulin resistance (del Rincon et al. 2007). The IGF system is also usually listed among the potential mediators of nutritional effects on folliculogenesis. The blood level of IGF-I is reduced during severe negative energy balance (Wathes et al. 2007) and increased during long-term positive energy balance, and thus the IGF system can convey metabolic information to the follicle. The action of IGF-I in the follicle is modulated by complex interactions with its receptor, the various IGF-binding proteins and IGF-II. Insulin-like growth factor I is a potent stimulator of follicular growth and follicular oestradiol secretion (Campbell et al., 1995; Campbell et al. 1996; Scaramuzzi et al.

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1999), and it is probably required for normal folliculogenesis and follicle selection (Mihm and Evans 2008). When used exogenously in the sheep auto-transplant model, it induced severe hyper-stimulation of folliculogenesis and follicular oestradiol and inhibin secretion (Scaramuzzi et al. 1999). Thus, under physiological conditions, the activity of IGF-I in the follicle is kept within precise limits by intra-follicular systems whose function is to prevent over-activity of follicular IGF-I. During negative energy balance, low IGF-I will inhibit folliculogenesis, but during positive energy balance, elevated IGF-I is likely to have only limited effects on the follicle because of intrafollicular inhibition by the IGFBPs and PAPP-A and sequestration of the IGF-IR by IGF-II.

Fatty Acids There is some evidence that unsaturated fatty acids also have a role in the regulation of female reproduction and more particularly in ovarian function. In cattle, dietary supplementation with various long-chain polyunsaturated fatty acids (PUFA), both n-3 and n-6, induced changes in oocyte quality and in several aspects of folliculogenesis. For example, long-chain PUFAs increased the total number of follicles and the size of the dominant or pre-ovulatory follicle (Staples et al. 1998). Several in vitro studies have investigated the effects of n3 and n-6 PUFAs on steroid production by bovine primary granulosa cells (Hinckley et al., 1996), and these data suggest that PUFAs either directly or indirectly exert differential effects on ovarian steroid synthesis. Peroxisome proliferator-activated receptors (PPARs) The PPARs are a family of three (a, b and c) ligandactivated transcription factors that share a common structure with steroid hormone receptors and are implicated in the regulation of energy balance and lipid metabolism, apparently acting as fuel sensors, providing cells with information about energy status. The endogenous ligands for PPARc are lipophilic compounds (Komar 2005) such as PUFAs, and eicosanoids derived directly from the diet or through metabolism (e.g., the prostaglandin D2 metabolite, 15-deoxy-12,14-prostanglandin J2, or PGJ2). Synthetic ligands for PPARc include the thiazolidinediones (TZDs), a class of insulinsensitizing agents (Forman et al. 1996). Deletion of the PPARa gene does not appear to affect the fertility of mice, whereas PPARb ⁄ d-null mice have placental malformations that are lethal to the conceptus early in pregnancy (Lee et al. 1995; Barak et al. 2002). In several species, all three PPAR isoforms have been detected in the ovary: PPARc is expressed strongly in granulosa cells and less so in theca cells and corpora lutea of rodents and ruminants (Dupont et al. 2008). In rodents, it was first detected early in folliculogenesis at the primary follicle stage, and its expression increased progressively to the ovulatory stage but decreased after the LH surge (Komar et al. 2001). Treatment with TZDs has produced contradictory effects on the secretion of ovarian steroids (inhibition or stimulation  2010 Blackwell Verlag GmbH

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of progesterone and oestradiol) in granulosa cells. They stimulate the in vitro secretion of progesterone and oestradiol by granulosa cells (Komar et al. 2001), whereas they inhibit the secretion of these steroids in human granulosa cells (Mu et al. 2000). In mice, the absence of PPARc in the ovaries reduced fertility, but there were no effects on folliculogenesis or ovulation rate; however, fewer embryos implanted, probably because of lower production of progesterone by the corpus luteum (Cui et al. 2002). Furthermore, if the deletion of PPARc was restricted to the mural granulosa cells of antral follicles, some follicles failed to rupture and the number of oocytes that ovulated was reduced (Kim et al. 2008). Overall, it seems highly likely that PPAR c has a role in folliculogenesis (Fig. 4).

FSH Signalling Pathways in Granulosa Cells The gonadotrophins exert their effects on the follicle acting through specific receptors belonging to the G protein class of trans-membrane receptors. Within the ovine follicle, granulosa cells express FSH receptors from the early pre-antral stage of folliculogenesis (Tisdall et al. 1995), while theca cells express LH receptors from the late pre-antral stage onwards (Logan et al., 2002). The granulosa cells of the potentially ovulatory follicle also express LH receptors (Carson et al. 1979). The gonadotrophins have stage-sensitive, multiple effects on folliculogenesis, thus FSH can stimulate granulosa cell proliferation and enhance cell survival, induce steroidogenic enzymes, alter the synthesis of extra cellular matrix components stimulate inhibin secretion, increase free calcium levels and regulate the activity of the intrafollicular IGF system. Cyclic adenosine 3¢,5¢-monophosphate (cAMP) was the first second messenger molecule to be identified for gonadotrophin action in the ovary. Following FSH binding to its receptor, cAMP is generated by adenylate cyclase and activates protein kinase A (PKA) by serine phosphorylation, PKA then phosphorylates the downstream substrate, cAMP response element-binding protein (CREB), resulting in gene transcription and synthesis of aromatase and inhibin. However, there are at least 100 genes that respond to FSH stimulation of

Fig. 4. Some mechanisms by which natural (fatty acids and prostaglandins) and synthetic (thiazolidinediones) peroxisome proliferator-activated receptors c (PPARc) ligands may function as nutrient sensors to modify follicular function. On ligand activation, PPARc dimerizes with RXRa to initiate gene transcription. PPARc = Peroxisome proliferator-activated receptor gamma; RXRa = retinoid X receptor alpha

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granulosa cells, and most are independent of CREB (Hunzicker-Dunn and Maizels 2006). In recent years, additional FSH-stimulated signalling pathways have been identified in granulosa cells. Follicle-stimulating hormone, activates the Phosphatidylinositol-3 kinase (PI3K) pathway in granulosa cells leading to the phosphorylation of several downstream pathways including the terminal kinases Akt and serum and glucocorticoid induced kinase (Gonzalez-Robayana et al. 2000). These terminal kinases have multiple substrates associated with protein synthesis, apoptosis and differentiation, and they appear to be critical for FSHinduced, granulosa cell differentiation and survival (Richards et al., 2002). The MAPK signalling pathway has at least three signalling cascades, all are present in granulosa cells and all appear to be responsive to FSH via PKA, activating the terminal kinases, extra cellular response kinase (ERK; Cottom et al. 2003), Jun N terminal kinase (JNK; Peter and Dhanasekaran 2003) and p38MAP kinase (Maizels et al. 1998) to influence a diverse range of FSH-stimulated functions such as steroidogenesis (Tosca et al. 2005), granulosa cell differentiation (Shiota et al. 2003), apoptosis (Shiota et al. 2003), oocyte maturation and germinal vesicle breakdown (Fan et al. 2004). Finally, FSH can also stimulate cAMP independent pathways such the PKC (Babu et al. 2000; Fan et al. 2004) and MAPK (Babu et al. 2000) resulting in cAMP-independent follicular responses to FSH.

Local Integrative Pathways and Mechanisms (Signalling Cross-talk) A concept not widely appreciated until recently, but now generally accepted, is that a cascade of multiple, divergent intracellular signalling pathways, that modify a wide array of cellular functions, is activated when gonadotrophins stimulate follicular cells (Richards et al., 2002; Hunzicker-Dunn and Maizels 2006). Importantly, insulin and IGF-I can also activate these pathways, except for the cAMP-PKA pathway. Thus, it is possible and even probable that insulin and IGF-I can modify many of the cells’ responses to FSH and vice versa. By replacing the ‘linear’ models of hormone

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Fig. 5. Sites of established (solid lines) and potential (dashed lines) cross-talk between the FSH (cyclic AMP-PKA) and insulin (ERK and PI3K) signalling pathways in granulosa cells and between the FSH signalling pathway and the hexosamine nutrient sensing pathway

signalling with functional interactions among signalling pathways (often referred to as ‘cross-talk’) and networks in the follicle (Taniguchi et al. 2006), we will improve our understanding of the physiological interactions among the inputs of insulin and the gonadotrophins. Details of insulin’s intra-follicular signalling pathways are now beginning to emerge, but knowledge of how insulin’s signalling pathways interact and influence gonadotrophin-stimulated steroidogenesis is speculative (Fig. 5). In recent studies, we have observed that a short-term nutritional stimulation with lupin grain increased granulosa cell IRS-2 and IRS-4 but reduced granulosa cell Aromatase (Somchit 2008). In another experiment, a short-term infusion of glucose at 10 mM ⁄ h increased the total number of follicles but decreased the level of phosphorylated Akt and AMPK as well as Aromatase in granulosa cell lysates (Gallet et al. 2009). The effects of these treatments on classical FSH signalling pathways and how and when these pathways interact in folliculogenesis remain to be explored. Acknowledgements Supported by the BBSRC (BB ⁄ 0018420 ⁄ 1) and the European Union Framework 6 funding (MEXC-CT-2006-042499). RJS is holds a Marie Curie Chair of Excellence (MEXC-CT-2006-042499).

Author contributions The authors contributed equally to the intellectual content of this paper

Conflicts of interest The authors declare that they have no conflicts of interest

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41 Zachow RJ, Magoffin DA, 1997: Direct intraovarian effects of leptin: impairment of the synergistic action of insulin-like growth factor-I on follicle-stimulating hormone-dependent estradiol-17beta production by rat ovarian granulosa cells. Endocrinology 138, 847–850. Zeleznik AJ, Saxena D, Little-Ihrig L, 2003: Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation. Endocrinology 144, 3985–3994. Submitted: 1 Feb 2010; Accepted: 23 Mar 2010 Author’s address (for correspondence): RJ Scaramuzzi, UMR Physiologie de la Reproduction et des Comportements, L’Institut National de la Reproduction, 37380 Nouzilly, France. E-mail: [email protected]