Foxo3 is a PI3K-dependent molecular switch controlling the initiation of oocyte growth.

In mammals, oocytes are packaged into compact structures-primordial follicles-which remain inert for prolonged intervals until individual follicles resume growth via a process known as primordial follicle activation. Here we show that the phosphoinositide 3-kinase (PI3K) signalling pathway controls primordial follicle activation through the forkhead transcription factor Foxo3. Within oocytes, Foxo3 is regulated by nucleocytoplasmic shuttling. Foxo3 is imported into the nucleus during primordial follicle assembly, and is exported upon activation. Oocyte-specific ablation of Pten resulted in PI3K-induced Akt activation, Foxo3 hyperphosphorylation, and Foxo3 nuclear export, thereby triggering primordial follicle activation, defining the steps by which the PI3K pathway and Foxo3 control this process. Inducible ablation of Pten and Foxo3 in adult oocytes using a new tool for genetic analysis of the germline, Vasa-Cre(ERT2), showed that this pathway functions throughout life. Thus, a principal physiologic role of the PI3K pathway is to control primordial follicle activation via Foxo3.

Loss of Lkb1 provokes highly invasive endometrial adenocarcinomas.

Mutations in the LKB1 tumor suppressor gene result in the Peutz-Jeghers syndrome, an autosomal dominant condition characterized by hamartomatous polyps of the gastrointestinal tract and a dramatically increased risk of epithelial malignancies at other sites, including the female reproductive tract. Here we show that female mice heterozygous for a null Lkb1 allele spontaneously develop highly invasive endometrial adenocarcinomas. To prove that these lesions were indeed due to Lkb1 inactivation, we introduced an adenoviral Cre vector into the uterine lumen of mice harboring a conditional allele of Lkb1. This endometrial-specific deletion of the Lkb1 gene provoked highly invasive and sometimes metastatic endometrial adenocarcinomas closely resembling those observed in Lkb1 heterozygotes. Tumors were extremely well differentiated and histopathologically distinctive and exhibited alterations in AMP-dependent kinase signaling. Although Lkb1 has been implicated in the establishment of cell polarity, and loss of polarity defines most endometrial cancers, Lkb1-driven endometrial cancers paradoxically exhibit (given their highly invasive phenotype) normal cell polarity and apical differentiation. In human endometrial cancers, Lkb1 expression was inversely correlated with tumor grade and stage, arguing that Lkb1 inactivation or down-regulation also contributes to endometrial cancer progression in women. This study shows that Lkb1 plays an important role in the malignant transformation of endometrium and that Lkb1 loss promotes a highly invasive phenotype.

Specificity of the requirement for Foxo3 in primordial follicle activation.

Primordial follicles are long-lived structures assembled early in life. The mechanisms that control the balance between the conservation and the activation of primordial follicles are critically important for fertility and dictate the onset of menopause. The forkhead transcription factor Foxo3 serves an essential role in these processes by suppressing the growth of primordial follicles, thereby preserving them until later in life. While other factors regulating primordial follicle growth have been described, most serve multiple functions at several stages of female germ cell or follicle development, and corresponding mouse mutants exhibit pleiotropic phenotypes with disruption of multiple stages of follicle assembly, development, or survival. To investigate the possibility that Foxo3 also functions in other aspects of ovarian development beyond its known role in primordial follicle activation (PFA), we performed detailed analyses of mouse ovaries including electron microscopy to study primordial follicle structure, assembly, and early growth. These analyses revealed that the timing of primordial follicle assembly, early oocyte survival, and the expression of early germ line markers were unaffected in early Foxo3 ovaries. Taken together, these studies demonstrate that the phenotype associated with Foxo3 deficiency is remarkably specific for PFA and further support the placement of Foxo3 in a unique phenotypic class among mammalian female sterile mutants. Lastly, we discuss the implications of the specificity of this mutant phenotype with regard to the hypothesis that oocyte regeneration may occur in adults and serves as a means to replenish oocytes lost via natural physiological processes.

Genomewide discovery and classification of candidate ovarian fertility genes in the mouse.

Female infertility syndromes are among the most prevalent chronic health disorders in women, but their genetic basis remains unknown because of uncertainty regarding the number and identity of ovarian factors controlling the assembly, preservation, and maturation of ovarian follicles. To systematically discover ovarian fertility genes en masse, we employed a mouse model (Foxo3) in which follicles are assembled normally but then undergo synchronous activation. We developed a microarray-based approach for the systematic discovery of tissue-specific genes and, by applying it to Foxo3 ovaries and other samples, defined a surprisingly large set of ovarian factors (n = 348, approximately 1% of the mouse genome). This set included the vast majority of known ovarian factors, 44% of which when mutated produce female sterility phenotypes, but most were novel. Comparative profiling of other tissues, including microdissected oocytes and somatic cells, revealed distinct gene classes and provided new insights into oogenesis and ovarian function, demonstrating the utility of our approach for tissue-specific gene discovery. This study will thus facilitate comprehensive analyses of follicle development, ovarian function, and female infertility.

Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre.

Cell type-specific genetic modification using the Cre/loxP system is a powerful tool for genetic analysis of distinct cell lineages. Because of the exquisite specificity of Vasa expression (confined to the germ cell lineage in invertebrate and vertebrate species), we hypothesized that a Vasa promoter-driven transgenic Cre line would prove useful for the germ cell lineage-specific inactivation of genes. Here we describe a transgenic mouse line, Vasa-Cre, where Cre is efficiently and specifically expressed in germ cells. Northern analysis showed that transgene expression was confined to the gonads. Cre-mediated recombination with the Rosa26-lacZ reporter was observed beginning at approximately e15, and was >95% efficient in male and female germ cells by birth. Although there was a potent maternal effect with some animals showing more widespread recombination, there was no ectopic activity in most adults. This Vasa-Cre transgenic line should thus prove useful for genetic analysis of diverse aspects of gametogenesis and as a general deletor line.