First published online May 23, 2008
doi: 10.1242/10.1242/dev.020560
Development 135, 2127-2137 (2008)
Published by The Company of Biologists 2008
Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in follicle development and ovulation
Heng-Yu Fan1,
Masayuki Shimada2,
Zhilin Liu1,
Nicola Cahill1,
Noritaka Noma2,
Yun Wu3,
Jan Gossen4 and
JoAnne S. Richards1,*
1 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX77030, USA.
2 Department of Applied Animal Science, Graduate School of Biosphere Science,
Hiroshima University, Higashi-Hiroshima, 739-8528, Japan.
3 Breast Cancer Center, Baylor College of Medicine, Houston, TX77030, USA.
4 NV Organon, part of the Schering-Plough Corporation, Target Discovery Oss,
Molenstraat 110, 5340 BH Oss, The Netherlands.
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Fig. 1. Conditional knock-in of KrasG12D in granulosa
cells. (A-F) In vivo recombination of the R26R locus in
ovaries by the Cyp19-Cre transgene. (A) ROSA26, (B)
ROSA26;Cyp19-Cre and (C) ROSA26;Cyp19-Cre, 48 hours after
eCG treatment. Images are of ovaries from 23-day-old mice showing β-gal
staining (blue). (D) Day10 and (E) Day23 without eCG, and (F) Day23 with eCG
treatment. Hematoxylin and Eosin staining of paraffin sections after
β-gal staining showing the expression of β-gal in the ovaries of the
ROSA26;Cyp19-Cre mouse. F, follicle; GC, granulosa cell. (G)
RT-PCR detection of KrasG12D and total Kras mRNAs
in LSL-KrasG12D;Amhr2-Cre and
LSL-L-rasG12D;Cyp19-Cre mouse ovaries. (H)
Immunofluorescence of KRAS in the ovary of a 6-week-old cycling wild-type
mouse.
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Fig. 2. Expression of KRASG12D in granulosa cells causes multiple
reproductive defects. (A) Continuous breeding assay showing the
cumulative number of progeny per female. The
LSL-KrasG12D;Amhr2-Cre and
LSL-KrasG12D;Cyp19-Cre females (n=6) were
subfertile. (B) Superovulation experiments showing that the ovulation
rate in response to gonadotropins was reduced in Kras mutant mice
(n=10) as compared with the wild type (WT). (C-J) Histology of
WT (C,E,G,I) and LSL-KrasG12D;Amhr2-Cre (D,F,H,J) ovaries
at 8 (C-F), 16 (G,H) and 48 (I,J) hours after hCG treatment. Histology of WT
(I) and LSL-KrasG12D;Amhr2-Cre (J) ovaries 48 hours after
hCG treatment shows that an oocyte is trapped in the corpus luteum of the
Kras mutant ovary (arrow).
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Fig. 3. KrasG12D conditional knock-in mice develop ovarian
lesions with altered granulosa cell proliferation, differentiation and
apoptosis. (A) Size differences in wild-type (WT) and
LSL-KrasG12D;Cyp19-Cre ovaries at various ages.
(B-D) Histology of WT (B) and
LSL-KrasG12D;Cyp19-Cre (C,D) ovaries at 6 months of age.
NF, normal follicle; AF, abnormal follicle. (E,F) BrdU
incorporation assay in 12-week-old WT (E) and
LSL-KrasG12D;Cyp19-Cre (F) ovaries. Abnormal follicle-like
structures are indicated by arrows (as below). (G,H)
Immunofluorescent detection of phospho-histone H3 (pHH3, red) in 12-week-old
WT (G) and LSL-KrasG12D;Cyp19-Cre (H) ovaries.
(I,J) BrdU incorporation (I) and immunofluorescence for the
mitosis marker phospho-histone H3 (J) indicate slightly increased levels of
proliferation in KrasG12D-expressing granulosa cells of
antral follicles, as compared with wild type. (K,L)
Immunohistochemical detection of PCNA in 12-week-old WT (K) and
LSL-KrasG12D;Cyp19-Cre (L) ovaries. (M,N)
Apoptosis assays in 4-week-old WT (M) and
LSL-KrasG12D;Cyp19-Cre (N) ovaries, 2 hours after hCG
treatment. (O,P) Immunofluorescent detection of cleaved caspase
3 (CC3) in 12-week-old WT (O) and LSL-KrasG12D;Cyp19-Cre
(P) ovaries.
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Fig. 4. KrasG12D downregulates genes essential for granulosa
cell differentiation and ovulation. (A) qRT-PCR of
ovulation-related genes from mouse whole ovary mRNAs. Six ovaries from
different animals were analyzed. (B) KrasG12D
downregulated the expression of Fshr and prevented the FSH-induced
expression of Lhcgr in cultured granulosa cells. Expression of
KrasG12D was induced by infecting the cells with an
adenoviral vector encoding Cre recombinase (Ad-Cre). FSH (100 ng/ml) was added
to the medium of cells infected, or not, with Ad-Cre. NT, non-treated.
Ppib was amplified by RT-PCR in the same samples, as loading
control.
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Fig. 5. KRASG12D activates the RAF1/MEK/ERK1/2 and PI3K/AKT pathways
in granulosa cells. (A) RAS activity in wild-type ovaries during
ovulation, as measured by a GST pull-down assay using RAF1 RAS-binding domain
(RBD) as the bait. NT, non-treated. (B) RAS-GTP levels increased in
immature LSL-KrasG12D;Amhr2-Cre and
LSL-KrasG12D;Cyp19-Cre ovaries, as measured by the GST
pull-down assay using both RAF1 RBD and p110 RBD. (C)
Phosphorylation of MEK1/2, ERK1/2 and AKT in wild-type and
LSL-KrasG12D;Cyp19-Cre ovaries after eCG/hCG treatment.
Total ERK1/2 and AKT are shown as loading controls. (D-F) Localization
of phospho-ERK1/2 in ovaries. The level of phospho-ERK1/2 was low in immature
wild-type mouse ovaries (D), but was increased in the large antral follicles 2
hours after hCG injection (E). By contrast, the phospho-ERK1/2 level remained
low in LSL-KrasG12D;Cyp19-Cre ovaries after the same
treatment (F). (G-I) Immunofluorescence of phospho-AKT in wild-type
ovaries. (G) Immature ovary; (H) 48 hours after eCG; (I) 2 hours after hCG.
(J-L) Immunofluorescence of phospho-AKT in
LSL-KrasG12D;Cyp19-Cre ovaries. (J) Immature ovary before
eCG treatment (abnormal follicle-like structures indicated by arrows); (K) 48
hours after eCG treatment; (L) 2 hours after hCG treatment. Three to six
ovaries from different animals of each genotype were analyzed in each of these
experiments.
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Fig. 6. Acute effect of KrasG12D expression in cultured
granulosa cells. (A) Expression of KrasG12D
mRNA in LSL-KrasG12D granulosa cells after infection with
Ad-CMV-Cre. (B) Levels of phospho-ERK1/2 and phospho-AKT
post-infection. (C) LSL-KrasG12D granulosa cells
infected with Ad-CMV-Cre and control vectors (Ad-CMV-GFP) for 48 hours were
stimulated with FSH, forskolin (Fo) or amphiregulin (AR) for 20 minutes. Each
agonist induced ERK1/2 phosphorylation in control granulosa cells, but the
responses were reduced in the cells expressing KRASG12D.
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Fig. 7. Mkp3 is a KRASG12D-induced gene involved in normal
and abnormal granulosa cell development. (A) Semi-quantitive RT-PCR
shows that Mkp3 mRNA levels were elevated in
LSL-KrasG12D;Amhr2- Cre ovaries as compared with those of
wild-type mice (n=3 for each genotype). (B) Mkp3 mRNA
expression was induced in wild-type granulosa cells by eCG/hCG treatment.
(C-H) In situ hybridization for Mkp3 in wild-type and
Kras mutant ovaries. Bright-field images show ovarian histology
(C,E,G), whereas dark-field images show the signals of Mkp3 antisense
probe (D,F,H). Mkp3 mRNA was detected in granulosa/cumulus cells at 4
hours after hCG (D), but not in immature wild-type ovaries (F). By contrast,
Mkp3 mRNA was detected in some preantral follicles (H, arrows) in
Kras mutant ovaries of the same age. (I,J)
Mkp3 siRNA decreased the Mkp3 mRNA levels in unstimulated
granulosa cells (I) or those stimulated with AREG (J). (K,L)
AREG induced transient phosphorylation of ERK1/2 in control granulosa cells;
however, in the granulosa cells treated with Mkp3 siRNA, levels of
phospho-ERK1/2 remained elevated for longer (K). (L) Intensity comparison of
phospho-ERK2/total ERK2. (M,N) PD98059 blocked AREG-induced
Mkp3 expression (M), when the ERK1/2 activation is blocked (N).
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Fig. 8. Schematic of the ovarian defects caused by the expression of
KRASG12D in developing follicles. In ovaries of wild-type mice,
the LH/hCG surge transiently activates RAS and its downstream effecters, the
ERK1/2 pathway and the PI3K pathway, which impact granulosa cell
differentiation and ovulation by regulating the expression of numerous genes.
ERK1/2 induces the expression of MKP3, which negatively regulates ERK1/2
activity in ovulating follicles as well as in cells expressing mutant
KrasG12D. PI3K regulates the phosphorylation of AKT and
FOXO1. When KRASG12D is expressed in the granulosa cells, it
interacts with both RAF1 and PI3K, activates ERK1/2 and AKT, respectively, and
leads to two major ovarian phenotypes. In small follicles, the granulosa cells
fail to differentiate and are devoid of their marker genes such as the FSH
receptor. Moreover, these granulosa cells are non-mitotic, non-apoptotic and
reside in abnormal follicle-like structures that accumulate in the ovaries of
the mutant mice. Those follicles that escape this senescent fate develop to
the antral stage but fail to ovulate because of the impaired expression of
genes associated with ovulation. In addition, the mutant antral follicles
exhibit reduced levels of phospho-ERK1/2 related to abnormally elevated levels
of Mkp3. Red lines, RAS-related events in normal ovaries; green
lines, KRASG12D-related events in mutant ovaries.
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© The Company of Biologists Ltd 2008
