Primordial follicle activation appears to be a stochastic process, but the need to ration a finite supply of primordial follicles throughout reproductive life suggests that this process is tightly regulated. We speculate that activation is influenced by positive and negative feedback loops, perhaps mediated by secreted, diffusible factors. Although such factors have not been definitively identified (Skinner, 2005
), our findings substantially advance our understanding of ovarian biology by demonstrating that such communication is integrated by Foxo3 and the PI3K signalling pathway acting within the oocyte itself (see for model of Foxo3 function within oocytes).
Fig. 7 Model of Foxo3 regulation by PI3K-Akt signaling pathway within oocytes in the control of primordial follicle activation. In resting state, absence of a presumptive ligand engaging receptor tyrosine kinase (RTK) complex or other cell surface receptor is (more ...)
Foxo1, Foxo3, and Foxo4 function similarly, are broadly expressed, and share partially redundant and overlapping functions, whereas Foxo6 is regulated differently and lacks a conserved Akt motif (van der Heide et al., 2005
). In genetic analyses of Foxo1, Foxo3, and Foxo4 in mice, a prominent tumor suppressor phenotype was not apparent in single gene knockouts, whereas post-natal inactivation of all three loci resulted in lymphomas and endothelial cell neoplasms (Paik et al., 2007
). Inactivation of two of three Foxo
genes gave rise to intermediate phenotypes, clearly demonstrating that the Foxos can compensate for one another, at least in some cell types. Given this potential for genetic redundancy, it was initially surprising that Foxo3 has a unique, non-redundant role in the suppression of primordial follicle activation. This may now be explained by our finding that Foxo3 appears to be the predominant Foxo protein in oocytes. It is also notable that at the protein level, Foxo3 is readily detectable only within oocytes, whereas at the mRNA level, Foxo3 is much more broadly expressed within the ovary (Castrillon et al., 2003
; Richards et al., 2002
). This observation suggests that post-transcriptional mechanisms regulating Foxo protein stability are highly significant in vivo. Consistent with this idea, Foxo protein stability has been found to be regulated by ubiquitylation and proteosomal targeting in a wide range of experimental model systems and cell types (Huang and Tindall, 2007
; Plas and Thompson, 2003
; Schisler et al., 2007
We discovered that within oocytes, Foxo3 is phosphorylated at an Akt site and that the phosphorylation status of this site tightly correlates with subcellular localization (cytoplasmic vs. nuclear) in vivo, arguing that Akt is the prime regulator of Foxo3 in the context of primordial follicle activation. Concordantly, oocyte ablation of Pten led to Akt hyperactivation, Foxo3 hyperphosphorylation, and export of Foxo3 from the nucleus to the cytoplasm, resulting in follicle activation. It is also possible that additional kinases or other types of post-translational modification contribute to the regulation of Foxo3 within oocytes, serving as additional layers of regulation, but our findings argue that such modifications are not the principal mechanism by which Foxo3 is regulated as a switch controlling primordial follicle activation.
The mTOR protein participates in two distinct complexes, mTORC1 and mTORC2, with different biological functions and specificities (Guertin and Sabatini, 2007). Our finding that oocyte Akt is phosphorylated at S473 argues that mTORC2 is active (or becomes activated) in primordial oocytes, since mTORC2 has recently been shown to be primary kinase regulating Akt via phosphorylation at this site. On the other hand, we found no evidence of increased phosphorylation of mTORC1 targets during follicle activation. Since at least some of these targets, including p-S6K (John et al., 2007
) and p-4EBP (unpublished data) become hyperphophosphorylated in more advanced follicles, mTORC1 activation may be important in promoting increased protein translation during oocyte growth, but not during activation per se.
Along these lines, it is notable that Pten
are genetically equivalent with regard to primordial follicle activation. Conditional deletion of both genes within oocytes using the Vasa-Cre
transgene led to nearly-identical phenotypes of global follicle activation. An even more striking demonstration of this genetic equivalence is that Vasa-Cre; PtenL/L
and Vasa-Cre; Foxo3L/L
females (as well as Foxo3−/−
females) are initially fertile (Castrillon et al., 2003
). Thus, neither Foxo3 nor Pten are essential for subsequent steps of follicle maturation, ovulation, fertilization, etc. These observations are further evidence that this pathway proceeds in a linear manner with Foxo3 downstream of Pten and PI3K/Akt, and that Foxo3 is the principal, if not sole output of this pathway. While the Foxos are known to be important effectors of PI3K/Akt signalling, we find it somewhat surprising that a single target, Foxo3, has such an overriding role as an effector of this pathway within oocytes, given the diversity of potential Akt substrates. It is also rather surprising that Pten inactivation and resultant Akt hyperactivation within oocytes does not disrupt subsequent steps of follicle maturation, ovulation, etc. Although it is likely that PI3K-Akt signalling participates in other aspects of oogenesis and follicle maturation, our findings demonstrate that this signalling pathway has evolved to serve a particularly critical role in primordial follicle activation.
Our investigations with Vasa-CreERT2
establish the feasibility of conditional gene targeting in primordial oocytes. This study represents the first example of conditional genetic inactivation in primordial oocytes in adult animals, and thus Vasa-CreERT2
should prove useful for a broad range of studies of oocyte activation, survival, and aging, as well as conditional genetic analysis of the male germ line (Fig. S2
). It is also remarkable that the efficiency of recombination was so high (90–95% of primordial oocytes), at least up to 6 weeks of age. Primordial follicles are mitotically inactive (Hirshfield, 1991
), and prior radiolabelling studies suggested that primordial oocytes are also metabolically quiescent (Bakken and McClanahan, 1978
; Hirshfield, 1991
; Moore and Lintern-Moore, 1974
; Moore et al., 1974
; Roversi and Silvestrini, 1963
). However, these prior studies employed methods of limited sensitivity. Since production of active β-galactosidase requires 1) tamoxifen-induced transport of Cre from the cytoplasm to the nucleus, 2) homologous recombination of loxP sites, 3) ATP-intensive processes including transcription, mRNA transport, and protein translation, the high efficiency of Cre-mediated recombination argues that primordial follicles are normally engaged in a surprisingly high degree of metabolic activity and turnover of cellular components. In these respects, our observations argue that primordial oocytes resemble neurons, a finding with intriguing implications for the age-associated oocyte loss that leads to the menopause. We speculate that oocytes may be subject to some of the same aging mechanisms that contribute to age-associated neurodegeneration, including oxidative stress and the accumulation of damaged or malfolded proteins (Mattson and Magnus, 2006
Lastly, our findings, including the demonstration that the pathway functions continually in adults and not just during a more limited stage of early development, further support the idea that defects in PI3K signalling may contribute to certain forms of female infertility due to primordial follicle depletion, such as premature ovarian failure and primary amenorrhea. These findings also raise the possibility that pharmacologic agents acting upon the PI3K pathway, several of which are in clinical trials (Granville et al., 2006
), may be useful in controlling follicle activation, treating infertility, or forestalling the menopause.