Our work began with our findings that the forkhead tanscription 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 the formation and maintenance of follicles and in germ cell survival, and are extending the studies to the comparative action of other master genes affecting follicle dynamics and the results of joint ablation of several of them. In anproject this last year, we have examined the effect of over-expression of Foxl2. We created transgenic mice over-expressing Foxl2 under the control of an Amhr2 promoter, active starting early in embryonic development. The transgene up-regulates genes downstream of Foxl2 in the ovary and down-regulates male-determining genes in the testis. A larger follicle pool was also seen in transgenic ovaries. By contrast, the maturation of follicles was normal, with the decrease of follicle numbers during aging proceeding at the same rate as in the wild-type. The over-expression of Foxl2 thus seems 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. 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, a lower expression of factors involved in follicle maturation, and increased fertility. 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. Furthermore, gene expression levels of markers for further development and aging are consistent with either a developmental delay or slower aging in transgenic ovaries. In addition, 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. Finally, we studied the fertility of aging mice up to 12 months of age. With aging and approach of menopause, transgenic females showed a greater fertility (31-49%) compared with wild-type animals. This is the first gain-of-function change in a gene that gives greater fertility throughout the reproductive lifespan, and suggests a possible point for pharmaceutical intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000647-15
Application #
8552429
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$755,138
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Marongiu, Mara; Deiana, Manila; Marcia, Loredana et al. (2016) Novel action of FOXL2 as mediator of Col1a2 gene autoregulation. Dev Biol 416:200-11
Pelosi, Emanuele; Forabosco, Antonino; Schlessinger, David (2015) Genetics of the ovarian reserve. Front Genet 6:308
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
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
Kashimada, Kenichi; Pelosi, Emanuele; Chen, Huijun et al. (2011) FOXL2 and BMP2 act cooperatively to regulate follistatin gene expression during ovarian development. Endocrinology 152:272-80

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