Menopause is the most sharply-defined features of female aging. Its genetic determination is clear from hereditary instances of premature ovarian failure (POF), a condition affecting 2-5% of female population and characterized by menopause before age 40. Our work discovered that the forkhead transcription factor 2 (Foxl2) is important in preventing POF;most strikingly, we showed that Foxl2 hereditary deficiency provokes POF in women with the Blephariphomisis-Ptosis-Epicanthus Inversus Syndrome (BPES). We have then shown over the last 6 years that Foxl2 is critical throughout female reproductive life, vitally involved in the ovary in somatic sex determination and maintenance as well as in the development and stabilization of ovarian follicles. The studies have led to a significant change in the paradigm for somatic sex determination and maintenance, from a formulation in which the ovary was a default pathway when the male-determining gene Sry was absent or inactive to a model in which Sry remains male-determinative but several other genes, and especially Foxl2, are actively required to determine ovary formation and stability, continually repressing the male pathway. We have continued to analyze the action of Foxl2 by generating mouse models in which Foxl2 is ablated or overexpressed, either alone or in combination with other important ovarian factors. We have now progressed further in the elucidation of the role of Foxl2 in sex determination and ovary development, as well as in functions in two other extra-ovarian tissues where it is expressed. In an extension of the studies of somatic sex determination, we demonstrated that Cyp26b1 and transcription factor genes Sf1 and Sox9 are co-expressed in Sertoli cells in mouse fetal testes, whereas Cyp26b1 and Sf1 are co-expressed in Leydig cells. Furthermore, qRT-PCR showed that Cyp26b1 up-regulation by Sox9/Sf1 was attenuated by Foxl2, whereas in Foxl2-null mice, Cyp26b1 expression in XX gonads was increased 20-fold relative to WT controls. These data support the hypothesis that Sox9 and Sf1 ensure the male fate of germ cells in part by up-regulating Cyp26b1, and that Foxl2 acts to antagonize Cyp26b1 expression (and Sox9 action) in ovaries. Another project has investigated the role of Foxl2 in follicle dynamics, specifically in their formation and maintenance. When Foxl2 is ablated primordial follicles fail to advance in folliculogenesis, leading to an ovary filled with immature follicles that will later degenerate. We were interested in finding out what the effect of an over-expression of Foxl2 would produce. We created transgenic mice over-expressing Foxl2 under the control of an Amhr2 promoter, active early in gonadal development. We confirmed that the transgene is expressed and up-regulates genes downstream of Foxl2 in the ovary, as well as down-regulaties male-determining genes in the testis. Oocyte/follicle counts in ovaries at several developmental stages (P0 P4 P21 and 3 6 9 12 months) are showing a systematically larger follicle pool in transgenic ovaries. By contrast, the maturation of follicles is normal, with the decrease of follicle numbers during aging proceeding at the same rate as in the wild-type, the over-expression of Foxl2 seem to have a primary effect on the initial formation of follicles. This finding is especially relevant to the role of Foxl2 in determining the ovarian reserve and thus, the age of menopause. We also investigated the role of bone morphogenetic protein 2 (Bmp2) and Foxl2 in the regulation of the activity of follistation (Fst, an autocrine glycoprotein involved in the control of follicle stimulation) in the mouse ovary. We found that Bmp2 and Foxl2 co-operate to allow for correct Fst expression throughout the developmental program of the ovary. Moreover, our study of another model, mice ablated for Wnt4 (another gene involved in somatic sex determination), showed reduced expression of Fst limited to early development, suggesting a role for Wnt4 in the initiation rather than the maintenance of Fst expression. Based on these findings, we proposed an inferred model in which the contribution of WNT4, FOXL2, and BMP2 to the regulation of Fst is likely to be sequential and stage-dependent. To initiate Fst expression, WNT4 appears to be the major regulator, whereas FOXL2 and BMP2 assume a more prominent role in maintenance of Fst expression. Foxl2 is also expressed secondarily in at least two extra-ovarian tissues, the pituitary and the eyelid. In the pituitary, we have found that Foxl2 is not required for the specification of gonadotropes, which secrete LH and FSH, the endocrine signals that regulate folliculogenesis in the ovary and spermatogenesis in the testis. However, in Foxl2 mutant mice FSH secretion is dramatically reduced and activin is unable to drive Fshb expression, suggesting that the mechanism of activin-dependent activation of Fshb transcription is impaired. A small number of gonadotropes in the ventromedial region of the pituitaries from Foxl2 mutant mice still maintain FSH expression, suggesting that an auxiliary independent mechanism independent of FoxL2- and activin can drive some Fshb transcription. Nevertheless, these data indicate that in addition to its role in the ovary, Foxl2 function is required in the pituitary for normal levels of expression of FSH. In the eye, we found Foxl2 to be involved in the Notch pathway in the formation of corneal and eyelid stroma in mice. We have described how the over-expression of Notch1 intracellular domain (N1-ICD) impaired eyelid levator smooth muscle formation by down-regulating Foxl2. This is similar to the effect of haplo-insufficiency of FOXL2 in humans, which results in. These data strongly imply that a physiologically low level of Notch1 is crucial for proper Foxl2 expression in periocular mesenchymal cells, which are in turn essential for normal eyelid development. In an independent line of research, we have investigated the role of another forkhead transcription factor, Foxo3, which is not involved in the formation of ovarian follicles, but acts to maintain them. Foxo3 is known from work in other systems to act in several biologic processes including cell cycle control, maturation, survival and apoptosis. In the ovary Foxo3 has a specific role in controlling the activation of primordial follicles, governing the size of the ovarian reserve. In its absence, others have shown that the entire cohort of primordial follicles activates and grows uncontrollably, leaving the ovary empty and female mice completely sterile by 15 weeks of age. We have now found that in young females the expression of a mutated form of Foxo3 that cannot be inactivated by phosphorylation causes a delay of follicular growth, with longer survival of primordial follicles and a slower decrease in the levels of markers that characteristically decline during ovarian aging. We also studied adult, premenopausal, and perimenopausal mice, and showed that when active Foxo3 is present, aging processes are slowed and the ovary retains a larger pool of primordial follicles. Numbers of follicles at all stages of folliculogenesis are much greater in the transgenic mice compared to wild-type. Furthermore, gene expression levels of markers for further development and aging are consistent with either a developmental delay or slower aging in transgenic ovaries. Finally we measured the production of gonadotropins, whose levels increase as menopause approaches (an assay method used clinically in humans to diagnose the onset of menopause). We found a higher level of gonadotropins in wild-type compared to age-matched transgenic mice, supporting the interpretation of slower aging in the transgenics.

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Barban, Nicola (see original citation for additional authors) (2016) Genome-wide analysis identifies 12 loci influencing human reproductive behavior. Nat Genet 48:1462-1472
Marongiu, Mara; Deiana, Manila; Marcia, Loredana et al. (2016) Novel action of FOXL2 as mediator of Col1a2 gene autoregulation. Dev Biol 416:200-211
Pelosi, Emanuele; Simonsick, Eleanor; Forabosco, Antonino et al. (2015) Dynamics of the ovarian reserve and impact of genetic and epidemiological factors on age of menopause. Biol Reprod 92:130
Marongiu, Mara; Marcia, Loredana; Pelosi, Emanuele et al. (2015) FOXL2 modulates cartilage, skeletal development and IGF1-dependent growth in mice. BMC Dev Biol 15:27
Lunetta, Kathryn L; Day, Felix R; Sulem, Patrick et al. (2015) Rare coding variants and X-linked loci associated with age at menarche. Nat Commun 6:7756
Pelosi, Emanuele; Forabosco, Antonino; Schlessinger, David (2015) Genetics of the ovarian reserve. Front Genet 6:308
Yamamizu, Kohei; Schlessinger, David; Ko, Minoru S H (2014) SOX9 accelerates ESC differentiation to three germ layer lineages by repressing SOX2 expression through P21 (WAF1/CIP1). Development 141:4254-66
Takasawa, Kei; Kashimada, Kenichi; Pelosi, Emanuele et al. (2014) FOXL2 transcriptionally represses Sf1 expression by antagonizing WT1 during ovarian development in mice. FASEB J 28:2020-8
Pelosi, Emanuele; Omari, Shakib; Michel, Marc et al. (2013) Constitutively active Foxo3 in oocytes preserves ovarian reserve in mice. Nat Commun 4:1843
Kashimada, Kenichi; Svingen, Terje; Feng, Chun-Wei et al. (2011) Antagonistic regulation of Cyp26b1 by transcription factors SOX9/SF1 and FOXL2 during gonadal development in mice. FASEB J 25:3561-9

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