Previous studies have equated FOXL2 as a crucial actor in the ovarian differentiation process in different vertebrate species. Its transcriptional extinction in the polled intersex syndrome (PIS) leads primarily to a drastic decrease of aromatase (CYP19) expression in the first steps of goat ovarian development. In this study, we provide a better characterization of early ovarian development in goat, and we provide experimental evidence demonstrating that FOXL2 represents a direct transcriptional activator of the CYP19 gene through its ovarian-specific promoter 2. Moreover, the ovarian location of FOXL2 and CYP19 proteins, together with their expression profiles in the female gonads, stress the involvement of FOXL2 co-factor(s) for regulating CYP19 transcription. Expressional analyses show that activin-A can be considered as a strong candidate for being one of these FOXL2 co-factors. Finally, we discuss evidence for a role of activin and estrogens in somatic and germinal cell proliferation occurring before germ cell meiosis. This period, of 20 days in goat, seems to have no equivalent in mouse. This species-specific difference could explain the phenotype discrepancy observed between XX goat PIS −/− and XX mouse Foxl2 −/− .

FOXL2 is a gene encoding a forkhead transcription factor, whose mutations are responsible for the blepharophimosis-ptosisepicanthus inversus syndrome that often involves premature ovarian failure. FOXL2 is one of the earliest ovarian markers and it offers, along with its targets, an excellent model to study ovarian development and function in normal and pathological conditions. We have recently shown that the aromatase gene is a target of FOXL2, and only three other targets have been reported so far. To detect potential transcriptional targets of FOXL2, we used DNA chips and quantitative PCR to compare the transcriptomes of granulosa-like cells overexpressing, or not, FOXL2. This analysis showed that mediators of inflammation, apoptotic and transcriptional regulators, genes involved in cholesterol metabolism, and genes encoding enzymes and transcription factors involved in reactive oxygen species detoxification were up-regulated. On the other hand, FOXL2 down-regulated the transcription of several genes involved in proteolysis and signal transduction and in transcription regulation. A bioinformatic analysis was conducted to discriminate between potential target promoters activated and repressed by FOXL2. In addition, the promoters of strongly activated genes were enriched in forkhead recognition sites, suggesting that these genes might be direct FOXL2 targets. Altogether, these results provide insight into the activity of FOXL2 and may help in understanding the mechanisms of pathogenesis of FOXL2 mutations if the targets prove to be the same in the ovary.forkhead ͉ infertility ͉ premature ovarian failure ͉ ovary

Polyalanine (polyAla) tract expansions have been associated with an increasing number of human diseases. Here, we have undertaken a functional study of the effects of polyAla expansions in the context of the transcription factor FOXL2, involved in cranio-facial and ovarian development. Using two cellular models, we show that FOXL2 polyAla expansions lead to protein mislocalization and aggregation in a length-dependent manner. The fraction of cells containing cytoplasmic staining displays a sigmoidal relationship with respect to the length of the polyAla tract, suggesting the existence of a threshold length above which protein mislocalization occurs. The existence of such a threshold might be rationalized if we consider that the longer the polyAla tract is, the higher its tendency to misfolding or to inducing spurious interactions with cytoplasmic components. To study the intranuclear dynamics of polyAla-expanded FOXL2, we performed fluorescence recovery after photobleaching experiments. The most unexpected result concerned the pathogenic protein containing 19 Ala residues in the run, which was virtually immobile, although this variant does not present a classical aggregation pattern. Luciferase assays and real time RT-PCR of many potential target genes showed that polyAla expansions induce different losses of activity according to the target promoters tested. We provide molecular explanations for these findings. Although our main focus is the mechanisms of pathogenesis of polyAla-expanded proteins, we discuss the potential relevance of polyAla length variation in micro- and macroevolution because polyAla-containing proteins tend to be transcription factors.