Conserved insulin signaling in the regulation of oocyte growth, development, and maturation

Molecular Reproduction and DevelopmentVolume 84, Issue 6 p. 444-459 REVIEW ARTICLEFree Access Conserved insulin signaling in the regulation of oocyte growth, development, and maturation Debabrata Das, Debabrata Das Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TexasSearch for more papers by this authorSwathi Arur, Corresponding Author Swathi Arur sarur@mdanderson.org orcid.org/0000-0002-6941-2711 Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas Correspondence Department of Genetics, Unit 1010, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Email: sarur@mdanderson.orgSearch for more papers by this author Debabrata Das, Debabrata Das Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TexasSearch for more papers by this authorSwathi Arur, Corresponding Author Swathi Arur sarur@mdanderson.org orcid.org/0000-0002-6941-2711 Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, Texas Correspondence Department of Genetics, Unit 1010, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Email: sarur@mdanderson.orgSearch for more papers by this author First published: 05 April 2017 https://doi.org/10.1002/mrd.22806Citations: 91AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Insulin signaling regulates various aspects of physiology, such as glucose homeostasis and aging, and is a key determinant of female reproduction in metazoans. That insulin signaling is crucial for female reproductive health is clear from clinical data linking hyperinsulinemic and hypoinsulinemic condition with certain types of ovarian dysfunction, such as altered steroidogenesis, polycystic ovary syndrome, and infertility. Thus, understanding the signaling mechanisms that underlie the control of insulin-mediated ovarian development is important for the accurate diagnosis of and intervention for female infertility. Studies of invertebrate and vertebrate model systems have revealed the molecular determinants that transduce insulin signaling as well as which biological processes are regulated by the insulin-signaling pathway. The molecular determinants of the insulin-signaling pathway, from the insulin receptor to its downstream signaling components, are structurally and functionally conserved across evolution, from worms to mammals—yet, physiological differences in signaling still exist. Insulin signaling acts cooperatively with gonadotropins in mammals and lower vertebrates to mediate various aspects of ovarian development, mainly owing to evolution of the endocrine system in vertebrates. In contrast, insulin signaling in Drosophila and Caenorhabditis elegans directly regulates oocyte growth and maturation. In this review, we compare and contrast insulin-mediated regulation of ovarian functions in mammals, lower vertebrates, C. elegans, and Drosophila, and highlight conserved signaling pathways and regulatory mechanisms in general while illustrating insulin's unique role in specific reproductive processes. Abbreviations cAMP cyclic adenosine 3′,5′-monophosphate CDK1 cyclin-dependent kinase 1 (also known as CDC2) ERK extracellular signal-regulated kinase FOXO Forkhead box "other" FSH follicle-stimulating hormone GVBD germinal vesicle breakdown IGF[1R] insulin-like growth factor [1 receptor] ILP insulin-like peptide IR insulin receptor LH luteinizing hormone MI/II meiotic division I/II MSP major sperm protein MPF maturation-promoting factor [m]TOR [mammalian] target of rapamycin PCOS polycystic ovary syndrome PI3K phosphatidylinositol-3-kinase PKA protein kinase A PTEN phosphatase and tensin homolog deleted on chromosome 10 rpS6 ribosomal protein S6 "[We] propose that insulin is a key, evolutionarily ancient regulator of female reproduction." 1 INTRODUCTION The successful propagation of a species requires proper oocyte development and ovarian function. Throughout the animal kingdom, oogenesis is regulated by a large number of intra- and extra-ovarian factors. In mammals as well as in other vertebrates, the follicle is the functional unit of the ovary, consisting of three cell types: an outer theca cell layer, an inner granulosa cell mass, and oocytes. Follicular development, meiotic resumption, and ovulation are orchestrated by the highly complex interplay of endocrine, paracrine, and autocrine signals. Three different tiers of regulatory organs—the hypothalamus, anterior pituitary gland, and gonads, the hypothalamic-pituitary-gonadal axis—regulate these events in the life cycle of an oocyte (Edson, Nagaraja, & Matzuk, 2009; Nagahama & Yamashita, 2008). Insulin, one of the most conserved molecules among animals, also regulates ovarian development and oogenesis. Insulin was discovered and initially characterized for its roles in regulating carbohydrate, fat, and protein metabolism in muscle, liver, and adipose tissues (Saltiel & Kahn, 2001). Recent studies from the last two decades, however, have rapidly expanded the activities that insulin participates in, including regulation of steroidogenesis in ovarian cells in vitro and in the stromal and follicular compartments of human and murine ovaries (Acevedo, Ding, & Smith, 2007; Poretsky, Cataldo, Rosenwaks, & Giudice, 1999). Underscoring these observations and the impact of insulin signaling on ovarian development is the observation that, in humans, hypo- or hyper-insulinemia is associated with significant alterations to ovarian function (Chang, Dale, & Moley, 2005; Colton, Pieper, & Downs, 2002; Diamanti-Kandarakis & Dunaif, 2012). Insulin receptor (IR)-dependent signaling also promotes oocyte maturation in lower vertebrates, such as Xenopus and zebrafish, wherein IR is expressed both in the thecal-granulosa compartment and the oocyte surface (Chuang et al., 1994, 1993; Das et al., 2016; Hainaut, Kowalski, Giorgetti, Baron, & Van Obberghen, 1991), and regulates oocyte development and maturation in flies and worms (Brown et al., 2008; Lopez et al., 2013; Wu & Brown, 2006). Together, these reports suggest that the role of insulin signaling during oogenesis and ovarian development is conserved from worms to humans, even though physiological and molecular differences exist in their pathways. The mechanisms regulating these diverse insulin-dependent functions are clearly of immense importance, as are their modes of execution throughout phylogeny. Potential interplay among these signaling events in oocytes undergoing meiotic progression is an area of active research. Keeping these developments in mind, we focus primarily on the recent advancements and the crucial role of insulin-induced signaling pathways in oocyte growth, development, and maturation. Our comparison of worms, flies, teleosts, and mammals highlights the evolutionary conservation of the insulin-signaling pathway in general, while illustrating its unique role(s) in specific reproductive processes. 2 INSULIN: AN EVOLUTIONARILY CONSERVED MOLECULE Insulin signaling regulates blood glucose levels and is essential for maintaining energy storage, glucose metabolism, glycogenesis, lipogenesis, cellular growth, survival, and reproduction—additionally it plays a role in aging (Kenyon, 2010; Poretsky et al., 1999; Taguchi & White, 2008; Tatar et al., 2001). Physiologically, the cellular function of insulin is primarily mediated by IR, a member of the receptor tyrosine kinase family that is expressed on the cell surface as a heterodimer of two identical α2β2 subunits (reviewed by Lawrence, McKern, & Ward, 2007). IR regulates two major cell signaling cascades that affect either metabolic or mitogenic functions: (i) the phosphatidylinositol-3-kinase (PI3K)/AKT and (ii) RAS/ERK (extracellular-signal regulated kinase) signaling pathways (Figure 1) (described in detail by Belfiore, Frasca, Pandini, Sciacca, & Vigneri, 2009; Cantley, 2002; Liao & Hung, 2010; Roux & Blenis, 2004; Taniguchi, Emanuelli, & Kahn, 2006). Figure 1Open in figure viewerPowerPoint Insulin-dependent signal transduction pathways. IR undergoes autophosphorylation and activation at the C-terminal domains upon ligand binding. Tyrosine-phosphorylated adaptor proteins recruit signaling proteins via specific protein domains. Active PI3K phosphorylates the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to produce the second messenger PIP3. Phospholipid phosphatases (e.g., phosphatase and tensin homolog deleted on chromosome 10 [PTEN] and SH2 domain-containing inositolphosphatase-2 [SHIP2]) dephosphorylate and convert PIP3 to phosphatidylinositol 4,5-bisphosphate, and negatively regulate the PI3K pathway. PIP3 recruits phosphoinositide-dependent kinase-1 (PDK1) and AKT to the plasma membrane, where PIP3-bound AKT is phosphorylated at Thr308 by PDK1 and Ser473 by Rictor-mTORC2. Activated AKT phosphorylates and regulates many target proteins (yellow and orange box[es]) either positively (→) or negatively (⊣) to mediate distinct biological events, including (i) maintenance of glucose homeostasis; (ii) cellular survival and growth; (iii) inhibition of apoptosis; (iv) angiogenesis; and (v) regulation of gene transcription and protein synthesis. AKT provides a direct link between insulin signaling and nutrient sensing. The ERK signaling pathway is triggered by activation of the small GTPase protein p21 RAS. Active diphosphorylated ERK shuttles between the cytoplasm and nucleus, and regulates substrate proteins in both compartments (green and orange boxes), to mediate cell growth, survival, and differentiation. 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; Casp9, Caspase9; DAPK, death-associated protein kinase; eIF4E, eukaryotic translation initiation factor 4E; eNOS, endothelial nitric oxide synthase; GSK3, glycogen synthase kinase 3; MDM2, mouse double minute 2 homolog; p90RSK, ribosomal protein S6 kinase; RHEB, RAS homolog enriched in brain; SHC, SH2 domain-containing; SOS, son of sevenless The components of insulin signaling, including ligands and receptor proteins, are highly conserved and active among metazoans (Figure 2). For example, Drosophila insulin-like peptides (ILPs) bind to and activate human IR, and can lower blood glucose levels in rats (Sajid et al., 2011). Reciprocally, bovine and human insulin can bind to Drosophila IR with moderate affinity (Petruzzelli, Herrera, Garcia-Arenas, & Rosen, 1985; Sajid et al., 2011), underscoring the structural and functional conservation of insulin signaling in these distant species. Additionally, insulin receptor substrates—PI3K, AKT, and RAS-ERK—are all regulated downstream of insulin signaling in mammals (Belfiore et al., 2009; Taguchi & White, 2008; Taniguchi et al., 2006; White, 2003), Drosophila (Badisco, Wielendaele, & Vanden Broeck, 2013; Claeys et al., 2002; Sim & Denlinger, 2013), and Caenorhabditis elegans (Kenyon, 2010; Murphy & Hu, 2013). Figure 2Open in figure viewerPowerPoint A conserved insulin-signaling pathway in the ovary. Activation of the IR leads to cellular signaling through pathways whose members have orthologs among all metazoans. The most conserved is the PI3K/AKT/FOXO pathway. The RAS/ERK signaling cascade downstream of insulin signaling is not yet documented in flies (blue box). Included with the well-characterized and conserved function of the insulin signaling pathway in mediating glucose homeostasis, metabolism, growth, is its regulation of reproduction, specifically oogenesis (see text for details) Members of the human insulin peptide family include insulin, insulin-like growth factor (IGF) 1 and 2, and seven relaxin-related peptides (reviewed by Fernandez & Torres-Aleman, 2012). Despite their similarities at the ligand and receptor levels, insulin regulates only metabolic effects, whereas IGFs act as potent growth and differentiation factors (Belfiore et al., 2009). Insulin is secreted from mammalian pancreatic β cells in response to glucose, while other nutrients, such as free fatty acids and amino acids, can augment glucose-induced insulin secretion (Fu, Gilbert, & Liu, 2013); much less is known about the regulation of insulin gene expression or insulin secretion from pancreatic β-cells of lower vertebrates. The transcription factors that regulate the expression of insulin in zebrafish, however, are homologous with those in the mammalian system (Biemar et al., 2001; Kinkel & Prince, 2009), prompting us to hypothesize that the regulation of gene expression in teleosts is similar among vertebrates, future research should help clarify these mechanisms. Mature insulin hormone is generated by cleavage of the preprohormone in vertebrates. Conversely, invertebrate genomes encode for small ILPs—although these ILPs do carry the dibasic cleavages sites, additionally, invertebrates express several proconvertases, suggesting that the ILPs maybe generated from longer peptides, much like cleavage of preprohormone in vertebrates. The Drosophila genome contains eight ILP-encoding genes (dilp1-8) (Brogiolo et al., 2001; Colombani, Andersen, & Leopold, 2012; Gronke, Clarke, Broughton, Andrews, & Partridge, 2010). These dilp-encoded peptides are structurally similar to preproinsulin, of which DILP2 has the highest homology (35% identity) with mammalian insulins (Brogiolo et al., 2001). The C. elegans genome similarly contains genes for ∼40 distinct ILPs (daf-28 and ins-1 through ins-39) (reviewed by Li & Kim, 2008). Ninety percent of C. elegans ILPs emanate from neurons that integrate environmental cues to signal to other cell and tissue types (Li, Kennedy, & Ruvkun, 2003; Pierce et al., 2001). In addition, ins-1, ins-7, ins-18, and daf-28 are expressed in non-neuronal tissues, such as the intestine (Li et al., 2003; Murphy, Lee, & Kenyon, 2007; Pierce et al., 2001). Systematic and genetic analysis of all 40 ILP genes in C. elegans as well as genetic analysis of the 8 dilp genes in Drosophila demonstrated that ILPs regulate crucial invertebrate physiology, either alone or in combination, and may possess functional redundancy and/or mediate feedback regulation (Cornils, Gloeck, Chen, Zhang, & Alcedo, 2011; Fernandez & Torres-Aleman, 2012; Gronke et al., 2010). Given the varied biological responses generated during the development of worms and flies in response to the different ILPs, important unanswered questions include if and how environmental or nutritional input specifically regulates the expression of each of the insulin ligand genes to generate the distinct biological responses. Work in Drosophila revealed that distinct ILPs are secreted in distinct manners. For example, DILP2, 3, and 5 are secreted by neurosecretory cells in the fly brain in response to distinct signals in larvae versus adults. Some of these mechanisms include glucose sensing (reviewed in detail by Nassel & Vanden Boreck, 2016). However, while IGF-binding peptides been identified in Drosophila (Honegger et al., 2008), whether or not their binding affinity varies with different ILPs in a tissue-specific manner and how they modulate the function of ILPs requires further investigation. 3 INSULIN REGULATION OF OOGENESIS AND OVARIAN DEVELOPMENT Oocytes are terminal gametes, thus their quality is the principal determinant of early embryonic survival, establishment and maintenance of pregnancy, fetal development, and the acquisition of several birth defects (Gilbert, Macaulay, & Robert, 2015; Zhang & Smith, 2015). The process of oocyte formation, growth, and development establishes the foundation of proper embryogenesis, and key events in oocyte development are shared among phylogenetically distant animal groups. For instance, oocytes originate from primordial germ cells during fetal development in almost all animals and actively migrate to the gonad during early embryonic development from across the gut; an exception is C. elegans, whose primordial germ cells do not need to migrate to the gonad, and instead are carried there during ventral ingression (reviewed by Richardson & Lehmann, 2010). In early growth phases, oocytes actively transcribe genes whose mRNA are then held in a translationally silent form, owing to shortened polyadenine tails (reviewed by Bachvarova, 1992; Mendez & Richter, 2001). These transcripts are stored for future oocyte development, such as during oocyte maturation (e.g., c-mos, cyclinB), and for post-fertilization processes that are required prior to the maternal-to-zygotic genome transition (reviewed by Langley, Smith, Stemple, & Harvey, 2014). Growing oocytes also take up amino acids and several macromolecules from the surrounding follicular cells. De novo synthesis and uptake of macromolecules are both essential for the growth, development, and maturation of the oocyte itself, as well as for the storage of information and materials necessary to support early embryonic development (Langley et al., 2014; Picton, Briggs, & Gosden, 1998; Sánchez & Smitz, 2012). Finally, oocytes of almost all metazoan species arrest at the diplotene (or diakinesis, in C. elegans) stage of prophase-I, known as the primary arrest point (Figure 3); this milestone further aids in the differentiation, growth, and development of oocytes (reviewed by Von Stetina & Orr-Weaver, 2011). Figure 3Open in figure viewerPowerPoint Stages of oocyte development and meiotic resumption. (a) Formation of primordial follicles varies by species. Primordial follicles develop in utero in humans, versus 1–2 days after birth in mice. A primary follicle, developed from the primordial follicle, consists of a prophase-I-arrested primary oocyte surrounded by somatic granulosa cells (GC). By the secondary stage, the primary oocytes grow, granulosa cells proliferate, and an additional layer of somatic thecal cells (TC) forms outside the basement membrane of the follicle. In both mice and humans, preantral follicle development does not require pituitary gonadotropins. At puberty, FSH secreted by the anterior pituitary promotes further granulosa cell proliferation and survival. As a fluid-filled antrum cavity (AC) begins to form, secondary follicles become early antral follicles. The full-grown primary oocyte is surrounded by proximal cumulus cells (CC) and distal mural granulosa cells in preovulatory follicles. After a LH surge, oocytes undergo meiotic maturation. (b) In fish, the oogonium proliferates and generates primary follicles, at which point the oocytes enter meiosis and arrest at diplotene. The oocytes then begin to enlarge, and form follicles. Next cortical alveoli accumulate within the oocytes, followed by vitellogenesis, which increases the size of the oocyte. Full-grown (FG) follicles initiate oocyte maturation. The germinal vesicle migrates from the center of the oocyte to the periphery, and breaks down in response to maturation-inducing steroid. (c) In Drosophila, the ovariole is composed of the germarium in the anterior-most part followed by a row of progressively older egg chambers. Within region 1 of the germarium, a cystoblast (CB), derived from a germ-line stem cell, divides four times via mitosis to form a 16-cell germ-line cyst (GCC). Meiosis begins at Stage 2A of the germarium, and the oocyte (Oo) arrests at diplotene of prophase-I. The posterior-most germ-line cell becomes the oocyte, whereas the remaining 15 cells become nurse cells (NC). The egg chamber is surrounded by a single layer of follicular cells (FC). At Stage 13, after an unknown developmental or hormonal signal, the oocyte resumes meiosis and progresses to MI. (d) In C. elegans, adult hermaphrodites have two U-shaped gonads containing germ cells arranged in a distal-to-proximal polarity with respect to the somatic distal tip cell (DTC) at the distal end and the proximal spermatheca/uterus. Germ cells proliferate mitotically at the distal end, known as the "mitotic zone." At the proximal end of the mitotic region, germ cells switch to meiotic prophase, where they are first in leptotene/zygotene (transition zone [TZ]) and then progress through an extended pachytene followed by diplotene and diakinesis around the loop region. Primary arrest of C. elegans oocytes occurs at diakinesis. In response to sperm and its secreted factor, MSP, the most proximal oocyte (−1) is induced to undergo meiotic maturation. The red and green triangles indicate prophase-I arrest and meiotic resumption, respectively 3.1 Mammalian oocyte growth, development, and maturation Gonadotropins (e.g., follicle-stimulating hormone [FSH] and luteinizing hormone [LH]) and sex steroids (e.g., estrogens and progesterone) regulate mammalian folliculogenesis in a stage-dependent manner. Early stages of follicle development—from primordial follicles until the preantral follicle stage—are gonadotropin-independent (Figure 3a) (Edson et al., 2009); instead, they are regulated by locally secreted ovarian factors, such as KIT ligand and its receptor (Nilsson & Skinner, 2004), nerve growth factor (Dissen, Romero, Hirshfield, & Ojeda, 2001), and members of the transforming growth-factor β superfamily, such as anti-Müllerian hormone (Durlinger et al., 2002, 1999), growth differentiation factor 9 (GDF9) (Elvin, Clark, Wang, Wolfman, & Matzuk, 1999), and bone morphogenetic protein 4 (BMP4) (Nilsson & Skinner, 2004), BMP7 (Lee, Otsuka, Moore, & Shimasaki, 2001), and BMP15 (Yan et al., 2001). Ovarian follicles respond to gonadotropins via their expression of functional FSH and LH receptors, which occurs from the preantral stages of follicular development until formation of a pre-ovulatory, Graafian follicle (Edson et al., 2009). Bidirectional signaling between oocytes and adjacent granulosa cells promote granulosa cell proliferation and differentiation by coordinating with FSH, which in turn drives granulosa cell survival, proliferation, and production of steroid hormones. IGF1 has synergistic and complementary functions in the development of the granulosa cells (Edson et al., 2009). Mammalian oocytes enter meiosis I during fetal development, and subsequently arrest at the diplotene stage of prophase-I until puberty (Sánchez & Smitz, 2012). High concentrations of intra-oocyte cyclic adenosine 3′,5′-monophosphate (cAMP) and activation of cAMP-dependent protein kinase A (PKA) are essential for the maintenance of this prophase-I arrest (Adhikari & Liu, 2014; Conti et al., 2002). Meiotic competence is acquired during later stage of folliculogenesis, and coincides with antrum formation (Figure 3a). Oocyte maturation induces the release of the oocyte from primary arrest, and enables progression through meiotic metaphase. In mammals, meiotic maturation is triggered by the preovulatory surge in LH via activation of cytosolic maturation promoting factor (MPF), a heterodimer of catalytic cyclin-dependent kinase 1 (CDK1) and its regulatory component Cyclin B (Adhikari & Liu, 2014; Nurse, 1990; Solc, Schultz, & Motlik, 2010). Active MPF promotes progression from meiotic metaphase I (MI) to metaphase II (MII) through Histone H1 kinase activation, chromosome condensation, spindle formation, dissolution of nuclear envelope (germinal vesicle break down [GVBD]), and release of the first polar body. 3.1.1 Role of insulin signaling in mammalian oocyte development Insulin is routinely used as a supplement for the in vitro culture of preantral follicles (Chen et al., 2015; Louhio, Hovatta, Sjöberg, & Tuuri, 2000); however, a role for insulin in regulating oocyte growth in vivo has yet to be documented. In vitro studies of cultured mammalian cells showed that insulin stimulation promotes oocyte growth by increasing either the number of gonadotropin receptors or the sensitivity and binding capacity of LH to its cognate receptor (Poretsky & Kalin, 1987). Insulin can also act synergistically with FSH to promote human ovarian thecal-interstitial cell differentiation and proliferation (Duleba, Spaczynski, Olive, & Behrman, 1997), as well as with LH/human chorionic gonadotropin to regulate ovarian function, given that experimentally induced hyperinsulinemia causes increased human chorionic gonadotropin-induced ovarian growth (Poretsky, Clemons, & Bogovich, 1992). These data suggest that insulin indirectly regulates folliculogenesis in later stages of oocyte development. Insulin also regulates ovarian androgen production in humans, suggesting it has an indirect role in early folliculogenesis since ovarian androgen is important for maintaining early follicular development. Indeed, the absence of ovarian androgen receptors in mice causes premature ovarian failure (Gleicher, Weghofer, & Barad, 2011; Shiina et al., 2006). Thus, the possibility of insulin regulation of early oocyte growth via androgen–androgen receptor system cannot be ruled out. In vitro culture-based experiments with early stages of human and non-human primate follicles suggest that insulin functions as a survival factor, as increased insulin levels reduce the quantity of atretic follicles, causing an overall increase in the number of viable follicles (Louhio et al., 2000; Xu et al., 2010). In goat preantral follicles, treatment with 10 ng/ml of insulin is associated with FSH-stimulated follicular development and survival (Chen et al., 2015). Taken together, these data implicate insulin function in multiple aspects of mammalian oocyte growth. 3.1.2 Role of PI3K/AKT and TOR signaling in mammalian oocyte growth and development PI3K/AKT pathways play a major role in the initial recruitment of primordial follicles into the growth phase, although the physiological regulators are not well defined. For example, over-activation of PI3K signaling by oocyte-specific deletion of murine Pten (Phosphatase and tensin homolog deleted on chromosome 10) causes premature activation of primordial follicles, resulting in their rapid depletion in the adult and premature ovarian failure (Reddy et al., 2008). Interestingly, loss of phosphoinositide-dependent kinase-1, a kinase upstream of AKT, in murine oocytes also leads to premature ovarian failure, but in a reciprocal manner (Reddy et al., 2009): deletion of Pdk1 causes premature ovarian failure as a result of accelerated, direct clearance of primordial follicles from their dormant state. These two types of premature ovarian failure, resulting from loss of Pten or Pdk1, represent distinct etiologies of the human counterpart. One key downstream mediator of the PI3K/AKT pathway is the transcription factor Forkhead box "Other" 3 (FOXO3). FOXO3 is phosphorylated, and thus inhibited, by activation of the PI3K pathway (Liao & Hung, 2010). Active FOXO3 blocks primordial follicle activation, likely via inhibition of the expression of genes essential to oogenesis and folliculogenesis (John, Shirley, Gallardo, & Castrillon, 2007). Mice lacking Foxo3 undergo premature activation of primordial follicles and further depletion of mature follicles (Castrillon, Miao, Kollipara, Horner, & DePinho, 2003). During oocyte development, PI3K signaling seems to function in line with Foxo3 since simultaneous depletion of Pten and Foxo3 in oocytes does not have a synergistic effect on follicle activation (Reddy et al., 2008). A constitutively active form of Foxo3 in murine oocytes, however, produces distinct phenotypes depending on the time and stage of the oocyte. For example, Liu et al. found that active FOXO3, when expressed via Zp3::Cre, causes infertility by blocking oocyte growth, follicular development, and ovulation (Liu et al., 2007). Pelosi et al. however, found that active FOXO3, when expressed via the Kit promoter, maintained an ovarian reserve and increased reproductive capacity in mice (Pelosi et al., 2013). Presumably these differences were due to the expression of FOXO3 at different stages of oocyte development: the Zp3 promoter is active at the primary stage of folliculogenesis and remains active in growing oocytes whereas the Kit promoter is expressed only in primordial and primary follicles (Pelosi et al., 2013). Thus, PI3K-dependent FOXO3 activity has different context-dependent outcomes during oocyte growth and development. Tuberous sclerosis complex (TSC) and mammalian target of rapamycin complex 1 (mTORC1) also regulate oocyte growth (reviewed by Adhikari & Liu, 2010). In mice, activation of mTORC1 by loss of either TSC1 or TSC2 activates dormant primordial follicles (Adhikari & Liu, 2010), presumably through elevated activity of ribosomal protein S6 (rpS6) kinase 1/rpS6 signaling, which promotes protein translation and ribosomal biogenesis in oocytes (Adhikari & Liu, 2010). These observations highlight the role of TSC/mTORC1 signaling in the regulation of female reproduction, although a role for mTOR signaling in mediating oocyte development adds layers of complexity. AKT, for example, regulates TSC/mTORC1 (Huang & Manning, 2009), and AKT-mediated phosphorylation of TSC2 disrupts the TSC1/TSC2 complex that, in turn, inhibits mTORC1, resulting in inhibition of oocyte growth (Huang & Manning, 2009). Inhibition of mTORC1 via deletion of Raptor, however, triggers compensatory activation of PI3K/AKT signaling that maintains normal ovarian follicular development and fertility (Gorre et al., 2014). Thus, the context of mTORC1 inhibition seems to be essential for mediating all the possible outcomes of oocyte growth. Interestingly, the net