Forkhead boxO Family Members Are Critical for Follicular Development and Luteinization.

The Forkhead boxO (Foxo) family of transcription factors is conserved from C.elegans to man and controls a wide range of cellular functions, including metabolism, cell survival and apoptosis in a cell and tissue specific manner. We have shown previously that Foxo1, Foxo3 and Foxo4 are differentially expressed in the various cell types of the mouse ovary. Foxo1 is highly expressed in granulosa cells and also oocytes (but not luteal cells) whereas Foxo3 is most abundant in oocytes but is also expressed in granulosa and luteal cells. Although these patterns of expression suggest that the FOXO factors serve important physiologic roles in granulosa cells, the critical role of Foxo3 in the maintenance of germ cell quiescence in primordial follicles, and the essential role of Foxo1 in embryologic development has significantly hindered comprehensive genetic analyses of these factors in somatic cells of the adult ovary. To overcome these limitations and gain new insights into the physiologic roles of the FOXO factors in granulosa cells, we generated Foxo1/3/4 tKO mice in which we conditionally inactivated all three Foxo genes in granulosa cells by mating mice expressing Foxo1/3/4 floxed alleles with mice expressing Amhr2-Cre. These matings have also generated the Foxo1/4 double KO and Foxo3 single KO mice. We document in Foxo1/3/4 tKO mice (by real-time RT-PCR and immuno-staining) that expression of each Foxo factor is reduced in granulosa cells whereas expression of Foxo1 and Foxo3 in oocytes remains intact. The Foxo1/3/4 tKO mice appear to be infertile based on preliminary breeding results of adult tKO females mated to fertile wild-type male mice and on the dramatic, consistent ovarian phenotype of tKO mice. Specifically, histological sections of ovaries of 2 month old tKO mice show that follicular growth and apoptosis are impaired and corpora lutea are absent. Granulosa cells within growing follicles exhibit abnormal detachment from the basal lamina but are not apoptotic. Immunohistochemical, real-time RT-PCR and in situ hybridization analyses of granulosa cell and luteal cell marker genes document that major changes in gene expression profiles occur in the Foxo mutant cells. Expression of the granulosa cell genes Inhba (activin), Cyp19a1 (aromatase), Bmp2 (BMP2), Nr5a1 (LRH1), Lhcgr (LH receptor) and the luteal cell gene Cyp11a1 (cholesterol side chain cleavage cytochrome P450) are altered dramatically both in vivo and in culture whereas expression of Fshr (FSH receptor) and Esr2 (estrogen receptor beta) remains unchanged. The Foxo1/4 and Foxo3 cKO mice are fertile indicating that there are overlapping and/or stage-specific functions of the Foxo factors in the ovary. These data indicate that FOXO factors impact granulosa cell fate decisions by regulating not only apoptosis but also other growth regulatory pathways. NIH-HD-16272 and -16229. (platform)