Developmental Biology 299 (2006) 1–11www.elsevier.com/locate/ydbio

Review

Control of mammalian oocyte growth and early follicular development by theoocyte PI3 kinase pathway: New roles for an old timer

Kui Liu a,⁎, Singareddy Rajareddy a, Lian Liu a,d, Krishna Jagarlamudi a, Karin Boman b,Gunnar Selstam c, Pradeep Reddy a

a Departments of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Swedenb Radiation Sciences, Umeå University, SE-901 87 Umeå, Swedenc Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden

d Qilu Hospital, Shandong University, Jinan, 250012, Shandong, China

Received for publication 13 November 2005; revised 1 June 2006; accepted 28 July 2006Available online 5 August 2006

Abstract

A large amount of information has accumulated over the past decade on how gonadotropins, steroid hormones and growth factors regulatedevelopment of the mammalian ovarian follicle. Moreover, the bi-directional communication between mammalian oocytes and their surroundingsomatic (granulosa) cells has also been shown to be crucial for this process. The intra-ovarian factors, or more specifically, the intra-oocytesignaling pathways that control oocyte growth and early follicular development are largely unknown, however. Based on both in vitro studies andin vivo functional studies using gene-modified mouse models, this review focuses on the key features of the phosphatidylinositol 3 kinase (PI3K)pathway in growing mouse oocytes and on the novel functions of the oocyte PI3K pathway in controlling mammalian oocyte growth and folliculardevelopment that have come to light only recently. We propose that the PI3K pathway in the oocyte, which is activated by granulosa cell-producedKit ligand (KL) via the oocyte-surface receptor Kit, may serve as an intra-oocyte network that regulates both oocyte growth and the earlydevelopment of ovarian follicles.© 2006 Elsevier Inc. All rights reserved.

Keywords: PI3K pathway; Mammalian oocyte growth; Early follicular development

Introduction: the mammalian ovary

The major function of the ovary is the differentiation andrelease of mature oocytes for fertilization, and thus successfulpropagation of the species. Additionally, the ovary producessteroid hormones that allow the development of femalesecondary sexual characteristics and also support pregnancy(McGee and Hsueh, 2000; Vanderhyden, 2002). In mammals,the ovary is a heterogeneous organ containing follicles andcorpora lutea at various stages of development. Each folliclecontains an oocyte that is surrounded by granulosa cells. Inorder to produce mature oocytes, follicles are activated from the

⁎ Corresponding author.E-mail address: kui.liu@medchem.umu.se (K. Liu).

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ydbio.2006.07.038

pool of dormant primordial follicles and develop throughprimary and secondary stages before acquiring an antral cavity.At the antral stage, most follicles undergo atretic degeneration,whereas a few of them grow further and reach the preovulatorystage under the cyclic gonadotropin stimulation that occurs afterpuberty (McGee and Hsueh, 2000). In response to preovulatorysurges of gonadotropin during each reproductive cycle, thedominant graafian follicle ovulates to release the mature oocytefor fertilization, whereas the remnant follicle undergoesluteinization to become the corpus luteum (for reviews, seeElvin and Matzuk, 1998; Matzuk et al., 2002; McGee andHsueh, 2000; Richards et al., 2002; Vanderhyden, 2002). Theprocesses of follicular activation, development, ovulation andluteinization have been shown to be dependent on numerousgenes, which was mostly revealed by using the gene knockoutmouse technology (Choi and Rajkovic, 2006; Matzuk et al.,

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2002; Matzuk and Lamb, 2002; Naz and Rajesh, 2005; Pangasand Rajkovic, 2006; Richards et al., 2002; Roy and Matzuk,2006).

Of the physiological processes in the mammalian ovary,oocyte development is less well understood because of the factthat oocyte growth occurs in a complex and changing milieu inwhich the surrounding somatic cells constantly undergoproliferation, differentiation and apoptosis, and also becauseof the practical difficulties of obtaining oocytes in adequateamounts. Even so, it is well known nowadays that the growthand maturation of oocytes require regulatory mechanismsinvolving both extrinsic and intrinsic signaling pathways(Kidder and Mhawi, 2002; McGee and Hsueh, 2000; Vander-hyden, 2002; Wassarman and Albertini, 1994).

Rapid oocyte growth during follicular activation and earlydevelopment

In mammals, oocytes develop from primordial germ cells(PGCs) which are first discernible in the extra-embryonicmesoderm (at day 7.2 (E7.2) in the mouse embryo) and migratethrough the hindgut and dorsal mesentery to colonize the genitalridge (Eppig et al., 2004; Ginsburg et al., 1990). Throughouttheir migration, PGCs undergo mitosis, which ceases aroundE13 in mice. The entire population then enters prophase ofmeiosis I (Eppig et al., 2004; McLaren and Southee, 1997) andin the mammalian perinatal ovary, oocytes that are arrested inthe diplotene stage of meiosis I become surrounded by a singlelayer of squamous somatic cells to form a finite population ofprimordial follicles (Eppig et al., 2004; Matzuk et al., 2002;Peters, 1969) (Fig. 1A). The resting primordial follicles arethought to serve as the source of developing follicles andoocytes (Eppig et al., 2004; McGee and Hsueh, 2000;Vanderhyden, 2002), although recent studies from the Tillylaboratory have suggested that postnatal oogenesis may alsooccur in female mammals (Johnson et al., 2004, 2005).

Fig. 1. Diagram of oocyte growth during follicular activation and early developmentfew flattened pre-granulosa cells. (B and C) During follicular activation and easimultaneously. The oocyte grows aggressively and undergoes an approximately 300-granulosa cells to 3 layers of cuboidal granulosa cells by the time the oocyte has alconsidered to take place in an FSH-independent fashion during follicular activatidevelopment, ovulation and luteinization are not shown.

One phenomenon worth noting during primordial follicleactivation and early development is the extremely rapid growthof the oocytes. During follicular activation and early develop-ment in mice, for example, the oocytes grow aggressively withan approximately 300-fold increase in volume during the 2- to3-week growth phase (Figs. 1A–C) (Wassarman and Albertini,1994), which is also accompanied by a 300-fold increase in RNAcontent (Sternlicht and Schultz, 1981; Wassarman and Albertini,1994) and a 38-fold increase in absolute rate of protein synthesisas calculated per hour and per oocyte (Schultz et al., 1979;Wassarman andAlbertini, 1994). These events are indicative of aperiod of robust cell growth with intense metabolic activity(Wassarman and Albertini, 1994). What is worth noting is thatalthough oocyte growth and granulosa cell proliferation belongto a coordinated course of development, these two processes donot take place simultaneously (Eppig et al., 2004; Wassarmanand Albertini, 1994). Oocytes complete most of their growthphase before the formation of a follicular antrum (Figs. 1A–C)(Wassarman and Albertini, 1994), and the increase in oocytediameter and volume during antral follicular growth is relativelysmall (Eppig, 2001; Eppig et al., 2004; Wassarman andAlbertini, 1994). During the phase of oocyte growth, granulosacells proliferate from one layer of a few flattened pre-granulosacells to three layers of cuboidal granulosa cells by the timeoocyte growth is almost complete (Figs. 1A–C) (Wassarman andAlbertini, 1994). Most of the granulosa cell proliferation anddifferentiation that is stimulated by follicle stimulating hormone(FSH) occurs after the oocyte has almost stopped growing(Eppig et al., 2004; Wassarman and Albertini, 1994).

Compared to the relatively good understanding of hormone-regulated follicular development, our knowledge of intra-ovarian or intra-oocyte factors that regulate oocyte growth andearly follicular development at the pre-FSH stage is limited(Elvin and Matzuk, 1998; Peters et al., 1975; Wassarman andAlbertini, 1994). A number of genes, mostly coding fortranscription factors such as FIGα (Soyal et al., 2000), Foxo3a

in mice. (A) A dormant mouse oocyte in a primordial follicle is surrounded by arly development, oocytes and granulosa cells grow in coordination, but notfold increase in volume. Granulosa cells only proliferate from a few flattened pre-most completed its growth. Oocyte growth and granulosa cell proliferation areon and early development. The subsequent processes of continued follicular

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(Castrillon et al., 2003), Nobox (Rajkovic et al., 2004), Foxl2(Schmidt et al., 2004; Uda et al., 2004), Sohlh1 (Pangas et al.,2006) and Sohlh2 (Ballow et al., in press) have been suggested tobe essential for regulating folliculogenesis.

Bi-directional communication between oocytes and theirsurrounding granulosa cells

Oocyte growth is dependent on signals, growth factors andnutrients, etc., from granulosa cells; at the same time, oocytes arenot only passive recipients but rather have key roles inmanipulating the proliferation and differentiation of granulosacells. It is common knowledge nowadays that bi-directionalcommunication between oocytes and somatic cells plays animportant role in ovarian follicular development (for somereviews, see Albertini and Barrett, 2003; Elvin and Matzuk,1998; Eppig, 2001; Eppig et al., 2004; Matzuk et al., 2002;McGee and Hsueh, 2000; Nilsson and Skinner, 2001; Shimasakiet al., 2004; Vanderhyden, 2002). Furthermore, it has beensuggested that the rate of follicular development is based on adevelopmental program intrinsic to the oocyte, and althoughgonadotropins are essential for driving the differentiation ofgranulosa cell phenotypes, the oocyte is probably the dominantfactor within its range of influence that determines the directionof differentiation and the function of the granulosa cellsassociated with it (Eppig, 2001; Eppig et al., 2002). The rolesof oocyte-derived factors such as growth and differentiationfactor-9 (GDF-9) and bonemorphogenetic protein-15 (BMP-15)in follicular development remain one of the most active researchareas in female reproduction (for some recent papers andreviews, see Hashimoto et al., 2005; Mazerbourg et al., 2004;McNatty et al., 2005a,b; Moore and Shimasaki, 2005; Pangasand Matzuk, 2005; Shimasaki et al., 2004; Thomas et al., 2005).

In spite of the paracrine interaction between oocytes andgranulosa cells which is possibly mediated by transzonalprojections (Albertini et al., 2001), oocytes are also incontinuous contact with granulosa cells through gap junctions.Gap junctions are intercellular plasma membrane channels thatallow direct inter-cytoplasmic movement of small moleculessuch as nutrients, ions and second messengers betweenneighboring cells (Kidder and Mhawi, 2002). The gap junctionsbetween oocytes and granulosa cells have been shown to bemediated by connexin 37, the expression of which is restricted tothe interface between oocytes and granulosa cells in folliclesfrom the primary stage onwards (Simon et al., 1997; Teilmann,2005; Veitch et al., 2004). Mice that are deficient in connexin 37fail to develop antral follicles, which is caused by the lack of gapjunction-mediated communication between oocytes and granu-losa cells (Kidder and Mhawi, 2002; Simon et al., 1997).

KL-Kit signaling during oocyte growth and early folliculardevelopment

The communication between oocytes and the surroundinggranulosa cells is largely dependent on the signaling between thereceptor protein tyrosine kinase Kit and its only known ligand,Kit ligand (KL), which is also referred to as stem cell factor

(SCF) or steel factor. Kit is expressed at the surface ofmammalian oocytes at all stages of follicular development inpostnatal ovaries of the mouse, the rat and humans (Driancourtet al., 2000; Horie et al., 1991; Manova et al., 1990; Orr-Urtregeret al., 1990), whereas KL is only produced by granulosa cells(Ismail et al., 1996; Laitinen et al., 1995; Manova et al., 1993;Vanderhyden, 2002). Several lines of evidence have indicatedthat growth of the ovarian follicle is dependent on KL-Kitsignaling at a time when functional FSH receptors are not yetexpressed in the mouse ovary (Albertini and Barrett, 2003;Driancourt et al., 2000; Eppig, 2001; Huang et al., 1993; Kurodaet al., 1988; Matzuk et al., 2002; Nilsson and Skinner, 2001;Packer et al., 1994; Parrott and Skinner, 1999; Vanderhyden,2002; Yoshida et al., 1997).

In a study by Packer et al. (1994), treatment of in vitrocultured follicles from 8-day-old mice (which containedgrowing oocytes) with KL resulted in a significantly increasedoocyte growth rate. In addition, a Kit antibody ACK2 thatinterferes with the binding of KL to Kit was found to severelyinhibit the growth of late fetal and neonatal oocytes in co-culturewith ovarian cells, but had less effect on partially grown oocyteswith diameters of 51–59.9 μm (Packer et al., 1994). It wastherefore suggested that KL and Kit are required to maintainoocyte growth in vitro (Packer et al., 1994). Similar results wereobtained with cultured postnatal ovaries from 4-day-old rats,where development of the primordial follicle was dramaticallyinduced by cultures with KL, but completely blocked by the Kitantibody ACK2, indicating that KL is necessary and sufficient toinduce primordial follicle development and initiate folliculogen-esis in the rat (Parrott and Skinner, 1999).

A convincing in vivo study by Yoshida et al. (1997) hasindicated the stepwise requirement for Kit in development of theovarian follicle in mice. In this study, postnatal mice at variousages were injected with the effective Kit-blocking antibodyACK2, and the first wave of synchronous follicular develop-ment was studied. The blockade of Kit signaling was found todisturb the onset of primordial follicle development, primaryfollicle growth and follicular fluid formation of preantralfollicles (Yoshida et al., 1997). On the other hand, primordialfollicle formation, ovulation and luteinization of the ovulatedfollicle were not affected by ACK2 (Yoshida et al., 1997). Theseresults suggest that the intra-oocyte KL-Kit interaction isimportant for early follicular development before any functionalFSH receptor is expressed in the mouse ovary and implies thatthe Kit-mediated signaling pathways in oocytes supportautonomous follicular development independently of gonado-tropins (Yoshida et al., 1997).

The phosphatidylinositol 3 kinase (PI3K) pathway

KL-Kit-mediated signaling cascades inside mammalianoocytes are largely unknown. The rapid oocyte growth duringfollicle activation and early development implies that thisprocess may require certain common pathways that are involvedin cell proliferation and survival, such as the PI3K pathway,which can be activated by Kit in somatic or cancer cells (Blume-Jensen and Hunter, 2001; Kissel et al., 2000). Kit can recruit

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PI3K from the cytoplasm to the cell membrane area through thebinding of the Src homology 2 (SH2) domain of the p85 subunitof PI3K to the phosphorylated tyrosine on Kit, and therebyactivates the PI3K (Blume-Jensen and Hunter, 2001; Cantley,2002; Stokoe, 2005) (Fig. 2). Thus, in mammalian oocytes, thePI3K pathway may be activated by cell-surface Kit.

Fig. 2. Receptor protein tyrosine kinase (RPTK)-triggered activation of the PI3 kina(p110) and a regulatory subunit (p85 in this figure). For various isoforms of catalytiHunter, 2001; Stokoe, 2005). Upon ligand binding, the RPTK molecules are inphosphorylated tyrosines that are capable of binding to the SH2 domain of the p85 subarea of the cell activates this lipid kinase and leads to the production of PIP3 from PIPIP3 through binding of their PH domains to PIP3, and Akt is subsequently phoconformation. The activated Akt is a serine/threonine kinase with various substrates.p27, which regulate cell survival and cell cycle entry. Also illustrated in the figure isphosphorylate Foxo3a and cause the cytoplasmic localization of this molecule. Wtranscription factor, leading to apoptosis and cell cycle arrest. Akt can directly phosphthe cytoplasm—whereby its inhibitory effect is abolished. The phosphorylation of Gglycogen synthesis. Akt also plays a critical role in cell growth by directly phosphtuberin (TSC2), which is an mTOR inhibitor. The molecules downstream of mTORprotein translation for cell growth. Note that this illustration is a simplified version ofkinases have not been shown due to space limitation (for detailed reviews, see Blum

The PI3K pathway is a “classic” signaling pathwayconsisting of various signaling molecules including kinases,phosphatases and transcription factors that establish cascades ofintra-cellular signaling that are fundamental for regulation ofcell proliferation, survival, migration and metabolism (Blume-Jensen and Hunter, 2001; Cantley, 2002; Stokoe, 2005). PI3Ks

se pathway. The class-I PI3Ks are heterodimers consisting of a catalytic subunitc subunits and regulatory subunits of PI3Ks, see references (Blume-Jensen andmost cases dimerized and autophosphorylated and present one or several

unit of PI3K. The recruitment of PI3K from the cytoplasm to the inner membraneP2. Kinases containing PH domains (including PDK-1 and Akt) are recruited bysphorylated by PDK-1 at threonine 308, which stabilizes Akt in its activatedSome of the Akt substrates are shown in this figure, including CREB, BAD andthe phosphorylation and regulation of Foxo3a, p27, GSK-3 and mTOR. Akt canhen unphosphorylated, Foxo3a will relocate to the nucleus and function as aorylate p27 at threonine 187 and trigger the shuttling of p27 from the nucleus toSK-3 by Akt leads to inactivation of this kinase, which will lead to increased

orylating mTOR, but more importantly by phosphorylation and inactivation ofare p70 S6 kinase and 4E binding protein 1 (4E-EP1), which directly controlPI3K pathway activation, and some of the regulation pathways and substrates ofe-Jensen and Hunter, 2001; Cantley, 2002; Stokoe, 2005).

Fig. 3. KL-regulated phosphorylation of Foxo1 and Foxo3a in growing mouseoocytes. Ovaries from 8-day-old postnatal mice were collected and oocytes(including those from the primordial and primary follicles) were isolated (Reddyet al., 2005), starved and stimulated with 150 ng/ml KL for 5, 15, 30 and 60 min,or pretreated with the specific PI3 kinase inhibitor LY 294002 (LY) for 1 hbefore treatment with KL for 15 min. The oocytes were then lysed and levels ofphosphorylated Foxo1 (Thr 24), phosphorylated Foxo3a (Thr 32) and totalFoxo1 were measured. The levels of total Foxo1 served as an internal control forequal aliquoting of oocytes and equal loading of the gel. It can be seen from thefigure that both Foxo1 and Foxo3a are rapidly phosphorylated upon stimulationby KL. The elevated phosphorylation levels of both molecules were diminished,however, if the oocytes were pretreated with LY 294002, indicating thatphosphorylation of Foxo1 and Foxo3a was achieved through the PI3K.

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themselves are lipid kinases that phosphorylate the 3′-OH groupof the inositol ring of inositol phospholipids. A phosphatase,PTEN (phosphatase and tensin homolog deleted on chromo-some ten), reverses this process and functions as a negativeregulator of PI3K (Fig. 2) (Cantley, 2002; Simpson and Parsons,2001; Stokoe, 2005). The phosphatidylinositol-3,4,5-triphos-phate (PIP3) can then recruit proteins containing lipid-bindingdomains (i.e., the FYVE and the pleckstrin-homology (PH)domains) from the cytoplasm (Pawson and Nash, 2000), such asthe serine/threonine kinases 3′-phosphoinositide-dependentkinase-1 (PDK-1) and Akt (also known as protein kinase B,PKB) (Fig. 2) (Blume-Jensen and Hunter, 2001; Cantley, 2002).Akt is a key molecule of the PI3K pathway. Upon activation,Akt not only regulates glycogen synthesis through phosphor-ylating and inactivating glycogen synthase kinase-3 (GSK-3)(Cross et al., 1995; Hajduch et al., 1998), but also regulates thecell cycle by preventing GSK-3β-mediated phosphorylationand degradation of cyclin D1 (Diehl et al., 1998) and bynegatively regulating the cyclin-dependent kinase (cdk)inhibitors p27kip1 (p27) (Blume-Jensen and Hunter, 2001;Shin et al., 2005; Viglietto et al., 2002) (Fig. 2). Amongvarious Akt substrates, the forkhead transcription factors of theFOXO family are given greater consideration in this review(see below). The FOXO factors are evolutionarily conserved,and three of the four members in this family, Foxo1 (FKHR),Foxo3a (FKHRL1) and Foxo4 (AFX), are substrates of Akt(Accili and Arden, 2004; Arden and Biggs, 2002). Inmammals, Akt can phosphorylate three conserved residuesof serine or threonine in Foxo1, Foxo3a and Foxo4, whichthen leads to their nuclear export and results in inhibition oftheir apoptotic transcriptional activities (Fig. 2) (Accili andArden, 2004; Arden and Biggs, 2002; Brunet et al., 1999;Nakae et al., 1999). Several in vitro studies have shown thatFOXO factors have important roles in mediating cell cyclearrest and apoptosis (Accili and Arden, 2004; Burgering andMedema, 2003; Tran et al., 2003).

Activation of the intra-oocyte PI3K pathway by KL-Kit ingrowing oocytes

The activation and function of the oocyte PI3K pathwayduring mammalian oocyte growth and early follicular develop-ment have not been addressed in detail until recently (Jin et al.,2005; Kissel et al., 2000; Liu et al., submitted for publication;Reddy et al., 2005). Components of the PI3K pathway,including Akt, Foxo3a (Reddy et al., 2005), GSK-3α andGSK-3β (Liu et al., in press), Foxo1 (Fig. 3) and p27 (Liu et al.,submitted for publication; Zhang et al., 1999) have been shownto be present in growing mouse oocytes. In addition, by using anin vitro oocyte culture system, it was shown that treatment ofcultured mouse and rat oocytes with KL can activate the PI3Kpathway (Reddy et al., 2005). Stimulation by KL not only leadsto a rapid phosphorylation of Akt (indicating activation), but atthe same time also leads to phosphorylation of Foxo3a andFoxo1 (indicating functional suppression) (Reddy et al., 2005,and Fig. 3). Regulation of the phosphorylation/activation statusof Akt, Foxo3a and Foxo1 by KL-Kit has been shown to be

achieved via PI3K itself, as pretreatment of cultured oocyteswith the PI3K-specific inhibitor LY 294002 was found to almostcompletely abolish the phosphorylation of Akt, Foxo3a andFoxo1 (Reddy et al., 2005; Fig. 3). The phosphorylation/inactivation status of another member of the PI3K pathway, theAkt substrate GSK-3 (including GSK-3α and GSK-3β), wasalso found to be regulated by KL in in vitro cultured mouseoocytes via activation of PI3K (Liu et al., in press). Thedownstream effectors of GSK-3 in growing mouse oocytes,however, have not been identified yet (Liu et al., in press).

The abovementioned data lead us to the important proposalthat the KL-Kit-activated intra-oocyte PI3K pathway may be ofimportance for oocyte growth and early follicular development.Given the fact that growing oocytes are a very special cellpopulation that is arrested in meiosis I, it is hard to generalizeabout the functions of the oocyte PI3K pathway from relatedstudies performed in other cell types. To date, information onthe functional roles of the oocyte PI3K pathway has mostlybeen obtained from gene-modified mouse models (see nextsection) and some in vitro studies.

Even if the main topic of this review is the PI3K pathway ingrowing oocytes, it is worth mentioning the supposeddependency of female germ cell survival on PI3K (Morita etal., 1999). In cultured fetal mouse ovaries, the combined actionsof KL, leukemia inhibitory factor (LIF) and insulin-like growthfactor-I (IGF-I) were found to be required for maximalinhibition of apoptosis in germ cells (Morita et al., 1999).During this process, the PI3K pathway has been shown to be anessential component of cytokine-mediated female germ cellsurvival, as the anti-apoptotic effects of KL/LIF or IGF-I werefound to be almost entirely eliminated by co-treatment of fetalovaries with either of two specific PI3K inhibitors, LY29400 orwortmannin (Morita et al., 1999). In another study with culturedovaries from newborn rats, KL was found to have a significantlypreventative effect on the apoptosis of oocytes, and this functionwas abolished by the PI3K inhibitor LY294002, indicating thatKL prevents oocyte apoptosis via the PI3K pathway (Jin et al.,2005). This result was further confirmed by data that LY294002

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could partially reverse KL-suppressed Bax expression and KL-enhanced Bcl-xL expression, suggesting that KL can preventapoptosis of oocytes by regulating the expression of Bcl-2family genes via the PI3K pathway of the oocyte (Jin et al.,2005).

Mutant mouse models indicating that the oocyte PI3Kpathway plays a part in early follicular development

Irrespective of the in vitro data that have accumulated, it isimportant to uncover the functions of the oocyte PI3K pathwayin the control of oocyte growth and early follicular develop-ment. At the same time, it is as important to identifydownstream effectors of the oocyte PI3K pathway in thesephysiological processes. Results obtained from in vitro culturescan be less informative due to possible artifacts resulting fromthe non-physiological nature of tissue/organ culture approaches.The role(s) of the PI3K pathway in the oocyte can beinvestigated more reliably or confirmed using gene-modifiedmice, including naturally occurring mutant mice, gene-deficient(knockout) mice and transgenic mice.

Mutant mice with spontaneously occurring mutations of theSl and W loci

In mice, KL and Kit are encoded by the steel (Sl) and do-minant white spotting (W) loci, respectively. Phenotypes of Sland W mutations suggest that KL-Kit signaling plays a part inmelanogenesis, hematopoiesis and gametogenesis (Russell,1979; Silvers, 1979). Many alleles of variable severity at boththe Sl and W loci having been described and characterized(Besmer, 1997). Historically, most of the spontaneouslyoccurring Sl and W mutations in mice have been found tocause deficiencies in gametogenesis, and sometimes absence ofgerm cells (Besmer, 1997; Besmer et al., 1993; Coulombre andRussell, 1954; Mintz and Russell, 1957). For example, in Slpan

female mice where the Sl locus contains a DNA rearrangementupstream of the coding region for KL that severely reduces thelevel of expression of KL, oocytes do not grow beyond adiameter of 22 μm, and postnatal folliculogenesis is arrested atthe primary follicle stage (Bedell et al., 1995; Huang et al.,1993). In addition, the Sld female mice exhibit infertility that iscaused by germ cell deficiency in the ovary (Brannan et al.,1991), and the Slcon and Slt mutant females have defects inpostnatal development of the ovarian follicle (Bedell et al.,1995; Kuroda et al., 1988). All of the data that are availablepoint to the idea that KL-Kit signaling is essential formaintenance of ovarian follicular development.

Mutation of PI3K binding tyrosine in Kit leads to arrest offollicular development and subfertility in mice

To specifically study the significance of Kit-activated PI3Kpathway in mice, a knock-in mouse model was generated wheretyrosine 719 (Y719) in the Kit molecule was mutated tophenylalanine (F) (Kissel et al., 2000). With this mutation, Kit isunable to bind to the p85 subunit of PI3K, resulting in failure of

PI3K pathway activation by the Kit molecule in mice, whereasactivation of other pathways by Kit is unaffected. Surprisingly,such mutant mice show no pigment deficiency or impairment ofsteady-state hematopoiesis. Gametogenesis, however, isaffected (Kissel et al., 2000). The homozygous mutant(KitY719F/KitY719F) male mice were found to be sterile, whichis caused by a block in spermatogenesis (Kissel et al., 2000). Inthe female KitY719F/KitY719F mice postnatal follicular develop-ment was delayed, with most of the follicles arrested at theprimary stage. The numbers of preantral and antral follicleswere greatly reduced due to impaired development of thefollicle; thus, the size of the ovary was also smaller. Theseresults demonstrate that the blockage of Kit-activated PI3Ksignaling in mice caused arrested follicular development at theprimary follicle stage (Kissel et al., 2000), thus providing keyevidence to link the Kit-activated intra-oocyte PI3K pathwaywith early follicular development. Superovulation in adultKitY719F/KitY719F female mice, however, produced low num-bers of ova, suggesting that the hormone-induced folliculardevelopment was not affected by the Y719→ F mutation in theKit molecule of mice (Kissel et al., 2000). This study indicatesthat it is the oocyte PI3K pathway activated by Kit – but notother pathways – that is indispensable for early folliculardevelopment in mice.

Knockout of Foxo3a leads to overactivation of primordialfollicles and overgrowth of oocytes in mice

As mentioned earlier, Foxo3a is a transcription factor thatmediates apoptosis and cell cycle arrest, and its activity ismanipulated by PI3K/Akt (Accili and Arden, 2004; Arden andBiggs, 2002; Brunet et al., 1999; Nakae et al., 1999). Thefunctions of Foxo3a in the control of oocyte growth andfollicular activation have attracted the attention of reproductivebiologists in recent years. In a pioneer study by Castrillon et al.(2003), mice deficient in Foxo3a were found to show interestingovarian phenotypes. Although no significant difference in theinitial pools of oocytes (at postnatal day 3) before follicularactivation was observed, the Foxo3a-deficient mice hadsubstantially elevated numbers of follicles at postnatal day 14,at 4.5 weeks and at 8.5 weeks. At 18 weeks of age, the knockoutmice were already devoid of viable follicles (Castrillon et al.,2003). Thus, it was concluded that in the absence of Foxo3a,primordial follicles undergo global activation into primaryfollicles, indicating that the PI3K pathway member Foxo3afunctions to repress activation of the primordial follicle.Moreover, oocytes in Foxo3a knockout mice are larger thannormal, suggesting that Foxo3a negatively regulates oocytegrowth (Castrillon et al., 2003). As expected, these miceshowed infertility after 15 weeks of age. The global activationof primordial follicles in Foxo3a knockout mice was furtherconfirmed by two other research groups who generated Foxo3a-deficient mice independently (Hosaka et al., 2004; Lin et al.,2004). Although the ovarian cell types or the developmentalstages at which Foxo3a functions to suppress follicularactivation were not addressed in the knockout studiesmentioned above, if we combine these reports with the

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information that Foxo3a is mainly expressed in the nuclei ofoocytes in the primordial and early primary follicles of mice(Liu et al., submitted for publication), it is clear that Foxo3a inthe oocyte is the negative regulator of oocyte growth andfollicular activation.

Expression of constitutively active Foxo3a in the oocyte leadsto retardation of oocyte growth and arrest of folliculardevelopment in mutant mice

In the mouse ovary, Foxo3a is mainly expressed in the nucleiof oocytes of primordial and early primary follicles, whereas itsexpression in oocytes of primary and developed follicles issignificantly downregulated (Liu et al., submitted for publica-tion). To investigate the physiological significance of thisdramatic downregulation of Foxo3a in oocytes of primary anddeveloped follicles, a transgenic mouse model was generatedwhereby constitutively active Foxo3a is maintained in oocytesusing the oocyte-specific ZP3 promoter. The expression of theconstantly active “negative”molecule Foxo3a in mouse oocyteswas found to cause retardation of oocyte growth, resulting in asignificant reduction in oocyte volume in secondary follicles.The transgenic mice also showed arrested follicular develop-ment. Thus, it is not strange that such female transgenic mice areinfertile (Liu et al., submitted for publication). Together withresults from the Foxo3a knockout reports, it is clear now that ofthe members of the oocyte PI3K pathway that may controloocyte growth and early follicular development, Foxo3a is akey molecule. The function of Foxo3a in oocytes does notappear to be compensated for by other FOXO family memberswhen knocked out in mice.

As a transcription factor, oocyte Foxo3a may serve to inhibitthe transcription of genes that are required for oocyte growthand follicular development, directly or indirectly. For example,in the BMP-15 promoter region there are several Foxo3abinding sites, suggesting that oocyte Foxo3a may negativelyregulate the transcription of BMP-15 directly. This hypothesis issupported by further mechanistic studies with the ZP3-Foxo3atransgenic mice, where the constitutively active Foxo3a inoocytes caused dramatic reductions in the expression of BMP-15, connexin 37 and connexin 43, all of which are known to beimportant for the establishment of paracrine and gap junctioncommunications in follicles (Liu et al., submitted for publica-tion). Accordingly, activation of ovarian Smad pathways (asmeasured by the phosphorylation states of Smad2 and Smad 1/5/8), which is important for the proliferation and differentiationof granulosa cells, was also dramatically suppressed in thetransgenic mice (Liu et al., submitted for publication). As aresult of the defects in oocyte-granulosa communicationmentioned above, the proliferation and differentiation ofgranulosa cells are dramatically arrested, and follicles of thetransgenic mice also respond poorly to exogenous PMSGtreatment due to very low expression of FSH receptor (Liu et al.,submitted for publilcation). In addition, the hindered oocyte-granulosa gap junction caused by reduced connexin 37 levelsmay lead to reduced transfer of nutrients, ions and secondarymessengers from granulosa cells to oocytes, which are required

for vigorous oocyte growth during early follicular development(Kidder and Mhawi, 2002; Vanderhyden, 2002). We suggestthat oocyte Foxo3a suppresses oocyte growth and folliculardevelopment through blockage of paracrine and gap junctioncommunications between oocytes and granulosa cells. Theresults above indicate that a well-balanced activation of theoocyte PI3K pathway is of great importance for a proper rate ofoocyte growth and follicular development.

Overgrowth of oocytes and overactivation of primordialfollicles in p27 knockout mice

One important downstream molecule of the PI3K pathway isthe cdk inhibitor p27, which is a negative regulator of themammalian cell cycle and cell growth (Fero et al., 1996). Byshuttling between the cytoplasm and the nucleus, the growth/cell cycle inhibitory function of p27 is prevented or initiated incells, respectively. It has been demonstrated that in several typesof cells this process is under the control of Akt and Foxo3a. Aktcan directly phosphorylate p27 at tyrosine 187 and trigger theshuttling of p27 from the nucleus to the cytoplasm—wherebyits inhibitory effect is abolished (Cunningham et al., 2004; Shinet al., 2005; Viglietto et al., 2002). Foxo3a has also been shownto regulate p27 expression, a process that is controlled by theupstream PI3K/Akt signaling (Chandramohan et al., 2004;Dijkers et al., 2000).

In the ovary, p27 is reported to be expressed in the nuclei ofoocytes of primordial, primary and secondary follicles (Zhang etal., 1999). In partially grown oocytes, however, the expression ofp27 was significantly decreased (Zhang et al., 1999), indicatingthat p27 within the oocyte may be a negative regulator of oocytegrowth. In spite of the well-known roles of p27 in follicularluteinization (Fero et al., 1996), we found recently that in p27knockout mice primordial follicles are overactivated, andoocytes are significantly enlarged as compared to wild-typemice (our unpublished data). These results implicate p27 asanother important downstream molecule of the PI3K pathwaythat negatively regulates oocyte growth and early folliculardevelopment. In accordance with this hypothesis, studies withthe ZP3-Foxo3a transgenic mice have shown that p27 expres-sion is constantly present in the nuclei of oocytes in thetransgenic mice, indicating that oocyte Foxo3a may facilitateprevention of shuttling of p27 from nucleus to cytoplasm, ormayprevent the downregulation of p27 expression in mouse oocytes(Liu et al., submitted for publication). Thus, Foxo3a and p27may function in coordination in primary oocytes to suppressprimordial follicles from being activated. The underlyingmechanism of how Foxo3a regulates p27, or vice versa, inmouse oocytes has not been revealed at this stage, however.

Akt, Foxo1 and GSK-3 mutant mice

In mammalian cells, three closely related isoforms of the Aktfamily have been identified and termed Akt1, Akt2 and Akt3.These proteins are products of three separate genes located ondistinct chromosomes and are widely expressed with a fewisoform-specific features (Tschopp et al., 2005). Mice deficient

8 K. Liu et al. / Developmental Biology 299 (2006) 1–11

in Akt1, Akt2 or Akt3 have been generated, but no studies offertility in Akt1 and Akt2 knockout mice have been reported(Cho et al., 2001a,b; Easton et al., 2005), and Akt3 knockoutmice have been reported to have normal fertility (Tschopp et al.,2005). Female Akt1-deficient females, however, have beenfound to show greatly reduced fertility, whereas the rate ofoocyte growth and early follicular development was not foundto be significantly different from that of wild-type mice whenthe first postnatal wave of synchronous follicular growth wasevaluated (our unpublished data). These results indicate that

Fig. 4. The oocyte PI3K cascade that regulates oocyte growth and early follicular doocyte. Dimers of KL bind to the Kit receptor and cause dimerization and activationincluding the phosphorylation of tyrosine 719 (Y719). The phosphorylated tyrosine 7subunit of PI3K, leading to the recruitment and activation of PI3K to the oocyte cellmany substrates of Akt, the phosphorylation of GSK-3, Foxo3a and Foxo1 (not shbecomes inactivated, and the physiological significance of the KL-inactivated GSK-3localization in the oocytes, preventing them from inducing apoptosis or cell cycle arrof oocyte growth and early follicular development—when constitutively active Foxoand Cx43 is dramatically reduced, which is thought to be the reason for the retarded ooPI3K pathway is believed to enhance oocyte growth and follicular development, probaBMP-15, but no direct evidence has been published to date. It is believed that a fine bapathway is required inside the oocyte for follicular activation and development to occthe cause of some human infertility diseases in which follicular development is hind

Akt1 may not be essential for early follicular development.Even so, this does not imply that the three Akt isoforms areunimportant for enhancement of oocyte growth, but rather, thatthe compensating roles of Akt2 and Akt3 in the absence of Akt1may explain why Akt1-deficient oocytes and follicles can stillgrow normally. Further studies using mice with combineddeficiencies of several Akt molecules, or using transgenicmouse models where Akt is overexpressed in oocytes, may helpin the study of the part each Akt plays in controlling oocytegrowth and early follicular development.

evelopment in mice. KL is produced by granulosa cells (GC) surrounding theof Kit, which leads to autophosphorylation and activation of the Kit molecule—19 (Y719-P) provides a docking site for the SH2 domains of the p85 regulatorymembrane. This then leads to the recruitment and activation of Akt. Among theown) have been demonstrated in mouse oocytes. The phosphorylated GSK-3is not clear at this stage. The phosphorylation of Foxo3a results in cytoplasmic

est-like processes. The oocyte Foxo3a has been shown to be a negative regulator3a is maintained in mouse oocytes, the production of BMP-15, connexin (Cx) 37cyte growth and arrested follicular development. On the other hand, the activatedbly due to the stimulated expression of oocyte growth factors such as GDF-9 andlance between the “positive”molecules and the “negative”molecules of the PI3Kur. Unbalanced activation or suppression of the PI3K pathway in oocytes may beered.

9K. Liu et al. / Developmental Biology 299 (2006) 1–11

Foxo1 deficiency in mice has been found to cause lethality inembryos (Hosaka et al., 2004), making it impossible to usethese mice to evaluate the function of Foxo1 in oocyte growth,unless an oocyte-specific Foxo1 conditional knockout isgenerated. GSK-3α and GSK-3β knock-in mice have alsobeen generated, where the Akt phosphorylation sites, serine 21of GSK-3α and serine 9 of GSK-3β, were mutated to alanine,which led to resistance to Akt inactivation in both molecules(McManus et al., 2005). However, no defect in femalereproduction or follicular development in the GSK-3 knock-inmice was reported, implying that mutations in Akt phosphor-ylation sites in GSK-3α and GSK-3β may not play anindispensable part in oocyte growth and follicular developmentin mice. It is also possible that Akt/GSK-3-mediated down-stream signaling is compensated for by other pathways inmouse oocytes. Thus, the significance of KL-Kit-PI3K-mediated GSK-3 inactivation in growing murine oocytes invitro remains elusive at this stage.

Summary and concluding remarks

This article has reviewed the novel part played by the intra-oocyte PI3K pathway in the control of mammalian oocytegrowth and early follicular development. Based on in vitrostudies as well as on in vivo results mostly obtained from gene-modified mouse models, an intra-oocyte network integrated bythe PI3K pathway has emerged, which is thought to beindispensable for oocyte growth and early follicular develop-ment in mice, and probably in other mammalian species too. Atheoretical signaling cascade, initiated by KL-activated oocytesurface Kit and followed by the subsequent activation of PI3Kby Kit, is illustrated in Fig. 4. Granulosa cell-derived KL bindsto and activates Kit, leading to dimerization, autophosphoryla-tion and activation of Kit receptor, which further activates PI3Kand Akt downstream. Akt may enhance oocyte growth and alsothe production of oocyte factors such as GDF-9 and BMP-15,which in turn stimulate the proliferation and differentiation ofthe surrounding granulosa cells. Akt can also phosphorylateGSK-3, which results in its inactivation, and phosphorylateFoxo3a, which leads to its cytoplasmic localization andsubsequent suppression of its function. The functionally activeFoxo3a in oocytes may suppress oocyte growth and secretion.As illustrated in Fig. 4, Foxo3a has been shown to be capable ofsuppressing the production of BMP-15, connexin 37, andconnexin 43 in mouse oocytes, which are important for oocyte-granulosa and granulosa-granulosa communications, and tofacilitate nuclear expression of p27 in the oocyte, which all leadto the arrest of oocyte growth and follicular development.

The PI3K pathway is a conventional signaling cascade. It hasbeen proven to have a set of versatile functions in manyphysiological and pathological processes (Blume-Jensen andHunter, 2001; Cantley, 2002; Stokoe, 2005). It is only in recentyears that the roles of this pathway in controlling mammalianoocyte growth and follicular development have been uncov-ered. Additional downstream effecters of PI3K, Akt andFoxo3a, etc., in growing mammalian oocytes remain to bediscovered. The uncovering of the involvement of the oocyte

PI3K pathway in oocyte growth and early follicular develop-ment in mice may help us to gain a better understanding ofmolecular mechanisms of some of the human infertility diseasescharacterized by defects in follicular development, such aspremature ovarian failure (POF). Thus, healthy and diseasedhuman ovary samples should be screened for the expressionlevels and localization patterns of the PI3K pathway members.

Acknowledgments

We apologize to those whose papers are not cited here due tolimited space: it does not mean that their work is any lesssignificant. This work was financially supported by the SwedishCancer Foundation (Project Nr. 4988-B04-01XAB), the Swed-ish Research Council (Project Nr. K2005-72X-15379-01A), theSwedish Cancer Research Foundation in Norrland (LP-04-1620;LP-05-1642), the Umeå University Biotechnology Grant and theMedical Faculty of Umeå University, Sweden.

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