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First published online 21 January 2004
doi: 10.1242/dev.00969

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The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance

Dirk Schmidt^*, Catherine E. Ovitt^*,, Katrin Anlag, Sandra Fehsenfeld, Lars Gredsted, Anna-Corina Treier and Mathias Treier

Developmental Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

View larger version (29K): [in a new window]   Fig. 1. Generation of a Foxl2lacZ allele by homologous recombination. (A) Genomic structure of Foxl2 locus showing the single coding exon (red box) containing the WH/forkhead domain (green box). The targeting vector contains a lacZ cassette (blue box) in frame with the first ATG and a loxP-flanked neomycin-resistance (pACN) cassette replacing sequences encoding amino acids 62-375 and part of the 3' untranslated region. A diphteria toxin (DTA) cassette was included for negative selection. A, Asp718; B, BsaBI; N1, NheI; N2, NcoI; S, SpeI; X, XhoI. (B) Southern blot analysis of genomic tail DNA probed with a 5'- and 3'-outside probe, respectively. Genomic DNA was digested with Asp718/SpeI. The 5' probe detects the expected 12.2 kb WT band and the 7.8 kb mutant band, whereas the 3' probe detects the 12.2 kb WT band and a 6.6 kb fragment after removal of the selection cassette. (C) Protein was extracted from P1 ovaries of WT and mutant mice and analysed by western blot analysis using the anti-Foxl2 antibody directed against the N-terminus of Foxl2. In WT extracts a doublet band was detected at around 50 kDa. This doublet band disappears in Foxl2 mutant ovary extracts. Instead the Foxl2LacZ fusion protein is detected at 125 kDa.

View larger version (84K): [in a new window]   Fig. 2. Foxl2lacZ expression during organogenesis of the ovary. ß-galactosidase staining of 12.5 dpc gonads (A,B), newborn (C,D) and adult ovaries (E,F) from Foxl2lacZ heterozygous animals. (A,B) Foxl2lacZ expression is sexually dimorphic. No expression is detected in male gonads, whereas Foxl2lacZ expression starts at around 12.5 dpc in female gonads. (C,D) In newborns Foxl2lacZ expression is detected in the somatic cells of the ovary. (E,F) In adult ovaries Foxl2lacZ expression is seen in granulosa cells with the highest levels in early follicles (black arrowheads), whereas expression declines during later stages of folliculogenesis (white arrowheads).

View larger version (40K): [in a new window]   Fig. 3. Maturation state of primordial follicles in WT and Foxl2lacZ homozygous mutant ovaries. (A) Foxl2lacZ expression determined by ß-galactosidase staining in newborn heterozygous and homozygous Foxl2lacZ ovaries. ß-galactosidase staining is more intense in Foxl2lacZ homozygous mutant ovaries than in Foxl2lacZ heterozygous ovaries because of the two lacZ alleles present showing that there is no somatic cell loss in mutant ovaries. The inset section shows a representative primordial follicle from a double immunofluorescence staining of a mutant newborn ovary with anti-lacZ (green) and anti-GCNA (red) antibodies. LacZ expression was only detected in somatic cells but not in oocytes. (B) Hematoxylin and Eosin staining of P1 WT and mutant ovaries. Formation of primordial follicles was observed in WT and mutant ovaries (black arrowhead marking squamous granulosa cells; white arrowhead marking oocyte). (C) Immunofluorescence for Gcna1 (red) and MSY2 (green) on WT and mutant newborn ovaries. Gcna1 is expressed in germ cells until the pachytene stage of the prophase of Meiosis I, whereas MSY2 expression starts at the diplotene stage of Meiosis I. Comparison of WT and mutant neonatal ovaries shows no apparent difference in the expression profile of the two proteins, providing evidence that primordial follicles have progressed through the prophase of Meiosis I. Corresponding pictures are photographed at the same magnification.

View larger version (104K): [in a new window]   Fig. 4. Representative histological overview of WT and Foxl2lacZ homozygous mutant ovaries at 2 weeks, 8 weeks and 16 weeks after birth (w, weeks). Overview of different stages of folliculogenesis in WT and mutant mice (A-I). (A) Two weeks after birth a cohort of secondary or preantral follicles with two layers of cuboidal granulosa cells have developed in WT ovaries (asterisks). (B) In contrast, only a few oocytes show substantial growth surrounded by a single layer of squamous-like granulosa cells close to the rete ovarii (asterisks) that never contained two layers of granulosa cells in Foxl2lacZ homozygous mutant ovaries. (C) Higher magnification of B; two advanced mutant follicles are shown where the oocytes are already undergoing atresia (black arrow, squamous-like granulosa cells; o, oocyte). (D) At 8 weeks of age, all stages of follicular development are seen in WT ovaries. (E) In contrast, despite the substantial growth of all oocytes in Foxl2lacZ homozygous mutant ovaries, widespread follicular atresia is observed. (F) Higher magnification of E; mutant follicles with large atretic oocytes are shown that are surrounded by only a single layer of squamous-like granulosa cells (black arrow, squamous-like granulosa cells; o, oocyte). (C,F) Granulosa cells did not complete the squamous to cuboidal transition which is particularly evident in the advanced follicles in 2- and 8-week-old Foxl2lacZ homozygous mutant ovaries. (G) At 16 weeks of age all stages of follicular development are still seen in WT ovaries. (H) At this stage the mutant ovary in Foxl2lacZ homozygous mutant mice is only one-twentieth the size of a WT ovary. (I) Only very few oocyte remnants (black arrow) and disorganized granulosa cells are retained in mutant ovaries. Corresponding pictures are photographed at the same magnification.

View larger version (112K): [in a new window]   Fig. 5. Proliferation status of WT and Foxl2lacZ homozygous mutant ovaries. (A-D) High magnifications of Hematoxylin and Eosin stainings showing WT and Foxl2lacZ homozygous mutant follicles 2 weeks and 8 weeks after birth. (A) Secondary follicle in WT ovaries 2 weeks after birth (black arrow, cuboidal granulosa cells; o, oocyte). (B) Primordial follicles in mutant ovaries 2 weeks after birth (white arrow, squamous granulosa cells; o, oocyte). (C) Preantral follicle in WT ovaries 8 weeks after birth (black arrow, cuboidal granulosa cells; o, oocyte). (D) Atretic follicles in mutant ovaries 8 weeks after birth (black arrow, squamous-like granulosa cells; o, oocyte). (E-H) Detection of apoptosis by TUNEL assay in WT and Foxl2-deficient follicles. (E,F) There is almost no apoptosis detectable in WT or in Foxl2lacZ homozygous mutant ovaries 2 weeks after birth. (G) An antral follicle in an 8-week-old WT ovary showing apoptotic nuclei in the granulosa cells. (H) Atretic follicles in 8-week-old mutant ovaries stain positive in the squamous-like granulosa cells. (I-L) Staining of control and Foxl2lacZ homozygous mutant ovaries with an anti-PCNA antibody. (I) Two weeks after birth granulosa cells in primary and secondary follicles stain positive for PCNA, whereas no staining is detectable in Foxl2lacZ homozygous mutant ovaries (J). (K) Positive PCNA staining in granulosa cells of an antral follicle in the WT ovaries. (L) In contrast, no staining was observed in the mutant ovary.

View larger version (124K): [in a new window]   Fig. 6. 35S-in situ hybridization analysis of Foxl2, activin-ßA, activin-ßB, follistatin, inhibin-, Amh (anti-Mullerian inhibiting hormone), Gdf9 and Kitl, at different stages of ovary organogenesis (P, postnatal day; w, weeks). (A) Foxl2 is expressed in granulosa cells at early stages of folliculogenesis, whereas its expression declines to low levels in adult mutant ovaries. (B) In 2-week-old ovaries activin-ßA is localized to granulosa cells of antral follicles, whereas it is absent in Foxl2-deficient ovaries. (C) In contrast, activin-ßB expression is unaltered in mutant ovaries compared with WT ovaries. (D) Expression of follistatin was significantly downregulated in Foxl2lacZ homozygous mutant ovaries after P1. (E) Expression of inhibin- was not affected in mutant ovaries. (F) The expression pattern of Amh, which is implicated in the activation of primordial follicles, exhibits significantly reduced expression levels in Foxl2lacZ homozygous mutant ovaries. (G) Gdf9 expression starts at the type 3a follicle stage in WT oocytes. In the 2-week-old Foxl2lacZ homozygous mutant ovary all oocytes express Gdf9, including regions that correspond histologically to Fig. 5B. (H) Kitl is expressed in pregranulosa and granulosa cells during folliculogenesis. In Foxl2lacZ homozygous mutant ovaries Kit ligand expression is maintained at all stages. At least three different ovaries were analysed per time point.

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