Apoptosis of Granulosa Cells and Female Infertility in Achondroplastic Mice Expressing Mutant Fibroblast Growth Factor Receptor 3G374R
Abraham Amsterdam,
Karuppiah Kannan,
David Givol,
Yoshio Yoshida,
Kimihisa Tajima and
Ada Dantes
Department of Molecular Cell Biology, Weizmann Institute of
Science, Rehovot 76100, Israel
Address all correspondence and requests for reprints to: Abraham Amsterdam, Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail:
abraham.amsterdam{at}weizmann.ac.il
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ABSTRACT
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Fibroblast growth factors play an important role in the control of
ovarian folliculogenesis, but the complete repertoire of ovarian
receptors which can transduce the fibroblast growth factor signals and
their precise localization in the ovary have not yet been
characterized. The most common form of inherited human dwarfism results
from a point mutation in the transmembrane region of fibroblast growth
factor receptor 3. A mouse model for achondroplasia was generated by
introducing the human mutation (glycine 380-arginine) into the mouse
fibroblast growth factor receptor 3 (G374R) by a "knock-in"
approach using gene targeting leading to a constitutively active
receptor. This resulted in the development of dwarf mice that share
many features with human achondroplasia. Here we report that female
(fibroblast growth factor receptor 3 G374R) dwarf mice become
infertile. While no significant changes were observed in the anatomical
and histological appearance of ovaries of 3-wk-old dwarf mice, a
dramatic difference was observed in ovaries of 3-month-old mice. The
normal ovary consists mainly of healthy corpora lutea and follicles at
different stages of development, whereas the ovaries of the dwarf mice
remain small and contain mainly follicles with a progressive apoptosis
in the granulosa cells, and no corpora lutea could be observed. The
levels of LH, FSH, and progesterone were lower by 72.3%, 38.0%, and
40.0%, respectively, in the blood of the dwarf mice compared with
normal mice, and the total bioactivity of pituitary FSH and LH was
lower by 65.6% and 79.6%, respectively, in the dwarf mice compared
with normal mice. However treatment with PMSG and human CG of the dwarf
mice led to rapid follicular development and formation of corpora
lutea. Interestingly, the expression of the tumor suppressor gene p53
was increased dramatically in ovaries of the dwarf mice. The presence
of the fibroblast growth factor receptor 3 cellular receptors in both
normal and dwarf animals was demonstrated by Western blot and
immunostaining. However, the distribution of the fibroblast growth
factor receptors in the two strains shows significant differences. In
the normal ovaries fibroblast growth factor receptor 3 was
homogeneously distributed on the cell membrane of the granulosa cells
and was absent in theca as well as corpora lutea cells, whereas in
dwarf mice ovaries it was highly clustered on granulosa cells and very
often appears in endocytic vesicles. Aged oocytes were more frequently
observed in preantral follicles of ovaries of the dwarf mice.
Nevertheless, oocytes isolated from antral follicles resume their
meiotic division at a high percentage, similar to oocytes obtained from
normal ovaries. The results imply fibroblast growth factor receptor 3
involvement in the control of follicular development through regulation
of granulosa cell growth and differentiation, and that unovulation in
the dwarf mice could be overcome in part by administration of exogenous
gonadotropins. Moreover, it is suggested that the infertile phenotype
is partially due to defects in the pituitary-gonadal axis.
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INTRODUCTION
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FIBROBLAST GROWTH FACTORS (FGFs) comprise
at least 19 mitogenic ligands (1, 2) which bind to four
high-affinity FGF receptors (FGFR14) and require heparan sulfate
proteoglycans for signaling through these receptors (3, 4). FGFs are implicated in diverse biological processes such as
cell growth, motility, and differentiation, angiogenesis, and wound
healing (5). Their role was also demonstrated in limb,
skeletal, lung, and brain development (6, 7).
FGFs also play an important role in follicular development and ovarian
function. FGF was found to be mitogenic in cultured bovine, porcine,
rabbit, guinea pig, and human granulosa cells and to delay their
differentiation (8, 9, 10). In porcine granulosa cells, basic
FGF (bFGF) suppresses induction of LH/CG receptors and progesterone
secretion. It suppresses FSH induction of LH receptor also in rat
granulosa cells in culture as well as aromatose activity
(11, 12, 13, 14) but enhances progesterone synthesis in
preovulatory rat granulosa cells alone and synergistically with FSH and
LH stimulation (15, 16). It was recently demonstrated that
the FGF effect on enhanced progesterone production is due to
up-regulation of the expression of the steroidogenic acute regulatory
protein, which facilitates the transfer of cholesterol into
mitochondria where conversion of cholesterol to pregnenolone take place
(16). Furthermore, bFGF stimulates tissue-type plasminogen
activation expression, PGE synthesis, and resumption of oocyte
maturation in rat follicles in vitro (17).
Thus, although FGF seems to delay the earlier steps of granulosa cell
differentiation stimulated by FSH, it seems to mimic some of the
ovulatory actions of LH (18).
Recently, more than 75 germ-line mutations at various domains of
FGFR13 were found to be responsible for various forms of skeletal
dysplasias, resulting in at least seven syndromes of craniofacial and
limb anomalies such as craniosynostosis and achondroplasia (reviewed in
Refs. 19, 20, 21). Various studies indicated that the common
cause of these phenotypes was the gain-of-function due to
ligand-independent activation of the mutant receptors (22, 23). The severity and location of the skeletal dysplasia was
correlated with the site of receptor expression and the extent of
activation of the mutant receptor (19, 21).
A notable example is the G380R mutation in the transmembrane region of
FGFR3, which is responsible for 98% of all cases of human
achondroplasia (24). To study this phenotype we generated
a mouse model for achondroplasia using gene targeting (knock-in) to
generate the mouse with mutant FGFR3G374R
(25). This mutant mouse strain was shown to share many
features with human achondroplasia. The achondroplastic phenotypes
develop after birth and begin to differ from their normal littermates 1
wk or 10 d after birth (25). Our analysis also showed
that some of the histological features in the growth plate of these
mice were more severe than those reported for human achondroplasia.
Surprisingly, the murine females were infertile whereas no female
infertility was reported in human achondroplasia.
In this paper we studied the changes in the ovary of the
achondroplastic female mice. We found that FGFR3 is expressed on the
membrane of the granulosa cells but not in the oocyte. The granulosa
cells of the mutant mice showed aggregation and endocytosis of the
mutant receptor accompanied by high incidence of apoptosis. These
results implicate FGFR3 in the regulation of granulosa cell
differentiation. The cause of infertility was in the failure of
follicular maturation, ovulation, and corpora lutea formation, due to
apoptosis of the granulosa cells, which could be overcome, at least in
part, by exogenous administration of gonadotropins to the dwarf
mice.
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RESULTS
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To investigate the differences between dwarf and normal mice,
ovaries from 3-wk- and 3-month-old mice were inspected. No significant
differences were found in the ovaries of 3-wk-old mice, which contained
primordial primary, preantral, and antral follicles (Fig. 1
). In contrast, striking differences
were observed in ovaries of 3-month-old female mice. While the mean
diameter of the 3-month ovary was 2.4 ± 0.35 mm, the mean
diameter of the dwarf mice ovaries was significantly smaller; 1.2
± 0.31 mm (n = 5) P < 0.01 (Figs. 2
and 3
).

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Figure 1. Sections of Ovaries Obtained from 3-wk-Old Normal
and Dwarf Female Mice
A, Normal ovary (Ov) is loaded with preantral and small antral
follicles. B, Higher magnification of part of the ovary showing
developed follicles and normal oocyte (arrows). C, Ovary
(Ov) of dwarf mice essentially in a similar size. D, Higher
magnification of part of the ovary showing atretic follicle with
condensed nuclei at the inner layers of the granulosa cells and in the
follicular antrum (bipolar twisted arrow). Two other
antral follicles appear normal (bipolar arrows).
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Figure 2. Gallery of Ovaries (Ov) of Normal and Dwarf Mice
Obtained from 3-Month-Old Female Sectioned Through the Center of the
Glands
Ovaries of dwarf mice were often removed with the fallopian tubes (T)
because of their smaller size. Their average diameter was 50% of the
normal ovary.
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Figure 3. Sections of Ovaries Obtained from 3-Month-Old
Normal and Dwarf Female Mice
A, Normal ovary loaded with corpora lutea (CL) and follicles at
different stages of development. B, Higher magnification of part of the
normal ovary showing two antral follicles (bipolar
arrow) and luteal tissue. C, Ovary of dwarf mouse demonstrating
small antral follicles at the periphery of the ovary. D, Higher
magnification of a part of the ovary with atretic follicles showing
condensed nuclei at the inner layers of granulosa cells of small antral
follicles (twisted arrows).
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The basal level of progesterone in the sera of 3-month-old normal and
dwarf animals was 30.15 ± 0.58 ng/ml (n = 6) and 18.16
± 0.60 ng/ml, respectively (40% reduction). The level of LH in the
normal mice was 1.80 ± 0.09 ng/ml while in dwarf mice it was
0.50 ± 0.10 ng/ml (73% reduction). The level of FSH in the
normal mice was 7.05 ± 0.40 ng/ml compared with 4.37 ± 0.30
ng/ml in the dwarf mice (38% reduction) (Fig. 4A
). To examine whether the reduction of
LH and FSH blood concentration is due, at least in part, to the
biosynthesis of these hormone we checked the total bioactivity of these
hormones in pituitary homogenates prepared from 3-month-old female
mice. While the bioactivity of LH and FSH was 2,275 ± 543 mIU
(mean ± SD, n = 6) and 607 ± 142 mIU
(mean ± SD, n = 6) per normal mouse pituitary
(equivalent to 159 ± 28 ng and 42 ± 9 ng of pure active LH
and FSH, respectively) the bioactivity of these hormones was 466
± 148 mIU for LH and 209 ± 92 mIU for FSH (mean ±
SD, n = 6) per dwarf mouse pituitary (equivalent to
33 ± 4 ng and 15 ± 3 ng of pure active LH and FSH,
respectively) (Fig. 5
). It should be
noted that while the total weight of 3-month dwarf mice decreased by
44.8% (27 ± 0.5 g vs. 14.9 ± 0.6 g),
the weight of the pituitary decreased by 59.3% (4.9±0.3 mg
vs. 1.9 ± 0.2 mg) with no change in the weight of the
hypothalamus (53 ± 3 mg vs. 56 ± 3 mg, n =
6) (Fig. 5
).

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Figure 4. Hormone Concentrations in Sera and Incidence of
Apoptosis in Ovaries of Normal vs. Dwarf Female Mice
A, Hormones in sera of 3-month-old normal and dwarf female mice.
Basal levels of progesterone, LH, and FSH were measured at the day of
diestrus in normal mice and at exactly the same day of age in dwarf
mice. Progesterone was assayed by RIA. LH and FSH were assayed for
their bioactivity using highly purified rat LH and FSH (iodination
grade) as standard and rLHR-15 and rFSH-17 responsive cell lines (see
Materials and Methods). Data are mean ±
SD (n = 6), a>b; c>d, and e>f,
P < 0.01. B, Incidence of apoptosis in preantral
and antral follicles of 3-wk- (3w) and 3-month (3 m)-old ovaries of
dwarf and normal mice. Data are mean ± SD of scoring
of apoptosis in sections of six ovaries doubly stained for DAPI and
TUNEL. c<d, P < 0.01. C, Apoptosis in follicles
of normal and dwarf mice stimulated with PMSG/hCG. Female mice (25 d
old) were injected with 15 IU PMSG, 48 h later with 15 IU/animal
of hCG. Animals were killed at 0 min time of injections (three
animals of each group) 48 h after PMSG injection and 48 h
after hCG injection. Data are mean ± SD obtained by
scoring of at least four sections of each gland doubly stained for DAPI
(DNA) and TUNEL (apoptosis). a>c and b>d, P <
0.01. m, Month; w, wk; D, dwarf; N, normal; hCG, human CG.
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Figure 5. Pituitary Gonadotropin Bioactivity and Body,
Pituitary, and Hypothalamic Weights of Normal vs. Dwarf
Female Mice
A, Bioactivity of LH and FSH and pituitary weight in normal and dwarf
3-month-old mice. LH and FSH bioactivity was monitored as specified in
legend to Fig. 3 and in Materiasl and Methods. Data are
calculated as mean ± SD per whole pituitaries (a>b,
P < 0.001; c>d, P < 0.001).
B, Relative weight of animals, pituitaries, and hypothalami. Original
data are expressed in grams or milligrams in Results.
Data are mean ± SD (n = 6). a'>b', P < 0.001;
c'>d', P < 0.001.
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While most of the ovarian tissue in the normal animals contained
corpora lutea and follicles at different stages of development, ovaries
of 3-month-old dwarf mice remained smaller in size, similar to ovaries
obtained from 3-wk-old animals. However, numerous follicles were
atretic with pyknotic nuclei (Fig. 3
).
The incidence of apoptosis was analyzed in ovarian sections of
3-month-old dwarf and normal mice by 4'6'diamido-2-phenylindole
hydrochloride (DAPI) and terminal deoxynucleotidyl transferase-mediated
nick end labeling (TUNEL) techniques
(Figs. 4
, 6
, and 7
). In the normal ovary of 3-month-old
mice some apoptosis could be detected, but most of the follicular
tissue did not show signs of apoptosis. In contrast, in ovaries of
3-month-old dwarf animals, condensed nuclei and large DNA aggregates
were evident mainly in antral follicles where most of the apoptotic
nuclei were confined to the inner follicular layers of the membrana
granulosa (Fig. 7
). Tissue sections stained for TUNEL showed
occasionally intensive labeling of fragmented DNA at the antrum of the
large follicles (data not shown), which suggests that the apoptotic
cells released their content into the follicular antrum. The incidence
of apoptosis was extremely low (<2%) in primordial and primary
follicles of 3-wk-old ovaries, which show normal oocytes with no
difference between normal and dwarf mice. The incidence of apoptosis
among large preantral and antral follicles of 3-wk-old normal ovaries
was 31 ± 0.7%, while that of dwarf mice of the same age was
33 ± 1.4% with no significant difference between these two
strains of mice (Fig. 4B
). In contrast, the incidence of apoptosis in
3-month-old normal ovaries was 32 ± 5.9%, while in the
dwarf mice it was 66 ± 1.7% (increase of 2.06-fold).

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Figure 6. Localization of FGFR3 in Normal and Dwarf Mice
A, DAPI staining and FGFR3 staining by immunocytochemistry of normal
follicle from ovary of 3-month-old female mouse. Nonapoptotic nuclei
delineating the organization of the follicular tissues. B, Staining of
FGFR3 in the some follicle with specific antibodies delineating the
surface of granulosa cells (arrow) with no labeling
within the oocyte (Oo) or theca cells (T). C, Apoptotic follicle
stained with DAPI. Note the fragmented DNA in nuclei of the inner
layers of granulosa cells and large spheres of DNA within the antrum
indicating intensive apoptosis (arrowheads). D, The same
follicle subsequent to staining with FGFR3 antibodies. Receptors are
located at the circumference of granulosa cells showing nonhomogenous
fragmented distribution of the receptor (arrow).
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Figure 7. TUNEL Staining and FGFR3 Localization in Ovaries
Obtained from 3-Month-Old Normal or Dwarf Mice
A, Note the absence of apoptotic granulosa cells (g) in the antral
follicle of a normal mouse. The labeling of the blood capillaries
within the theca (T) is not specific. B, Localization of FGFR3 at the
circumference of granulosa cells (arrows). C, TUNEL
labeling of an antral follicle of a dwarf mouse. Note the labeling at
the inner layers of granulosa cells (arrowheads). D,
Fragmented labeling of FGFR3 on granulosa cell surface and in endocytic
vesicles (twisted arrows).
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Ovarian tissue sections were also stained with anti-FGFR3 antibodies
(Figs. 6
and 7
). In ovaries of normal 3-month-old mice, positive
staining was evident on the circumference of granulosa cells. The
staining was found to be homogeneously distributed at the cell
circumference, but oocytes were devoid of specific staining. No
staining was detected in theca and corpora lutea cells. In dwarf mice
the labeling was also confined to the granulosa cells. However,
staining with anti-FGFR3 of the cell membrane was not homogenous but
showed aggregates and patches. In atretic follicles internalization of
the receptors in endocytic vesicles was clearly evident. Immunoblots of
ovarian homogenates clearly show the expression of FGFR3 both in normal
and dwarf mice (Fig. 8A
). The receptor
also shows an intensive band in brain homogenate, which serves as a
positive control, and also in liver and spleen, while only weak
staining was found in the adrenal (not shown). Intensity of staining of
the FGFR3 in Western blot was essentially the same in normal and dwarf
mice. We also examined the expression of oncogenes and tumor suppressor
genes by gel electrophoresis followed by Western blots. While there
were no differences in MDM2 expression, there was a dramatic elevation
of p53 expression in ovary, lung, and spleen of dwarf mice (Fig. 8
, B
and C), which may suggest that modulation of p53 expression may play an
important role in the high apoptotic incidence in the ovaries.

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Figure 8. Western Blot of FGFR3, p53, and MDM2
A, The receptor is expressed in normal and dwarf mice (3-month-old) in
the brain, ovary, lung, and spleen. B, High expression of p53 in dwarf
ovary, lung, spleen, and immortalized granulosa cell line (HO23)
expressing the temperature-sensitive mutant of p53 (Val 135 p53), which
serves as a positive control (37 ). C, No difference in
expression of Mdm2 in normal and dwarf mice.
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We intended to examine whether infertility and the absence of
ovulation in dwarf mice was due to the inability of follicles to
reach the Graafian follicle stage or to the inability of the oocytes to
resume their meiotic division. Ovaries of 3-month-old mice were
punctured with a syringe needle, and oocytes from both dwarf and normal
ovaries were released (six ovaries for each group) and incubated in
isotonic solutions at 37 C for 1224 h. The vast majority of oocytes
from both normal (87 ± 2.8%) and dwarf mice (85 ± 3.1%)
resumed their meiosis at the same rate as observed by the extrusion of
the first polar body (Fig. 9
, A and B),
indicating that the infertility of the dwarf mice is due to failure in
follicular maturation rather than to a defect in oocyte maturation.

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Figure 9. Oocytes Removed from Antral Follicles of
3-Month-Old Normal Female Mouse (A) and 3-Month-Old Dwarf Mouse (B)
Note extrusion of the first polar bodies after 24 h of incubation
of the oocytes in vitro. Cumulus cells are still
surrounding the oocyte.
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To examine whether the failure in follicular development and enhanced
apoptosis in dwarf mice are due to deficiencies in circulating
gonadotropins, we injected 25-d-old female of both dwarf and normal
mice with 15 IU of PMSG. Forty-eight hours later 15 IU of hCG were
injected. The ovaries of both strains were examined by histology and
TUNEL method at 0 time after injection of PMSG and at 0 and 48 h
after injection of hCG. This hormonal treatment was reported to
accelerate follicular development and sexual maturation leading to
massive ovulation (26, 27). The 25-d-old ovaries of the
dwarf mice were smaller (72% in diameter) than those of the normal
mice and consisted mainly of large preantral follicles and a few antral
follicles (Fig. 10
, A and B). The
interstitial tissue in the normal ovaries was more developed compared
with the dwarf mice ovaries. Forty-eight hours after PMSG injection,
there was a dramatic increase in the development of the normal mice
ovaries as expected, the diameter of which increased by 54%. The
ovaries contained many large preovulatory follicles (Fig. 10C
).
Similarly, there was also a dramatic development of the dwarf mice
ovaries (61% enlargement in diameter), and the ovaries consisted
mainly of large preantral follicles and some antral follicles (Fig. 10D
). The incidence of apoptosis was reduced significantly [from
48 ± 2% and 47 ± 2.6% to 20 ± 4.3% and 18 ±
2.4%, respectively (P < 0.01)] for normal and dwarf
ovaries (Fig. 4C
). After 48 h of hCG injection the normal ovaries
contained about six corpora lutea per ovary with no further significant
increase in size (Fig. 10E
). On the other side, dwarf ovaries continued
to develop (15.5% increase in the mean diameter, P < 0.01) containing
a large number of antral follicles, and one to two corpora lutea (Fig. 10F
). The incidence of apoptosis remained low in both strains after
PMSG and hCG treatment (Fig. 4C
). Therefore, it seems that gonadotropin
treatment of the dwarf mice can repair, at least in part, the
development of the ovary by reducing the apoptotic incidence and
accelerating follicular maturation and ovulation.

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Figure 10. Stimulation of 25-d-Old Female Normal and Dwarf
Mice with PMSG and hCG
Females (25 d old) were injected with 15 IU of PMSG for 48 h
followed by 15 IU of hCG, and animals were killed 48 h after hCG
injection and ovaries were processed for histology. A, Section of ovary
of 25-d-old normal mouse. Note preantral and small antral follicles,
which occupy most of the peripheral ovarian tissue of normal ovary. B,
Section of ovary of 25-d-old dwarf mouse. Note the smaller size of the
ovary (70% to that of the control in diameter), which mainly consists
of preantral and some small antral follicles. C, Development of the
normal ovary after PMSG injection (15 IU PMSG for 48 h), which
contains mainly large antral and preovulatory follicles. D, Development
of the dwarf mouse ovary 48 h after PMSG injection, which contains
mainly large preantral and medium sized antral follicles. E, Normal
ovary showing massive ovulation and formation of corpora lutea (*)
after 48 h of PMSG (15 IU) injection and 48 h after hCG
injection. F, Pronounced increase in the size of the antral follicle in
dwarf mice ovary after the same hormonal treatment. One corpus luteum
is visible in the ovarian section (*).
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DISCUSSION
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Previous studies demonstrated the expression of FGFR1, FGFR2, and
FGFR4 in the ovary (28, 29). Here, we have shown that in
mouse ovary FGFR3 is expressed in the granulosa cells but not in the
theca cells or the oocyte. Using immunostaining with antibodies to
FGFR3, the receptor was found to be localized to the membrane of the
granulosa cells. On the other hand, in granulosa cells of dwarf mice
containing FGFR3G374R, aggregation and
internalization of the receptor were observed.
Activation of the receptors by FGF leads to clustering and
internalization followed by receptor degradation or recirculation.
Therefore, it is possible that constant activation of the mutant
receptors even without ligand may lead to intensive internalization.
The role of FGFR3 in ovarian development is still obscure, but our
results suggest that it plays a role in the maturation of the follicle
via the function of the granulosa cells. This conclusion can be drawn
since granulosa cells expressing FGFR3G374R
undergo apoptosis, and at 3 months of age the ovary of the dwarf mice
showed many immature follicles and lack of Graafian follicles. On the
other hand, the oocytes in the ovaries of the mutant mice did not show
any defect and when released from the immature follicles could complete
the meiotic cycle similar to oocytes from wild-type mice. An
alternative possibility, which should be taken in consideration, is
that the entire phenotype of the ovary described in the present work
may be indirect and result from changes in pituitary function, namely
chronic low levels of FSH and LH that cannot support complete
follicular development or luteinization. Pituitary insufficiency
clearly could lead to apoptosis. Moreover, as demonstrated in this
study, the failure in follicular growth and luteinization can be
recovered to a large extent upon PMSG/hCG treatment. These observations
would strengthen the notion of an endocrine effect. An additional
possibility is that the ovarian phenotype leading to infertility in
achondroplastic mice expressing mutant FGFR3G374R
may be due to the small size of the mice.
The effect of the mutant FGFR3 on granulosa cells should be viewed in
light of recent studies on the signaling pathways of FGFR3 in various
cell types. These studies demonstrated that FGFR3 signaling could
follow three different pathways, depending on cell type and stage, as
described in the following examples. First, in fibroblasts the
signaling by FGFR3 leads to cell division, and this may be considered
as the proliferative or mitogenic function of receptor activation which
operates through the FRS, ras, and MAPK pathway (30).
Second, in chondrocytes, signaling by FGFR3 probably functions through
the signal transducer and activator of transcription (STAT)
pathway and transcription activation of cyclin-dependent
kinase (cdk) inhibitors (e.g.
p21waf or p16), which lead to cell growth arrest
(31, 32). Indeed, FGFR3-deficient mice showed bone
overgrowth (33, 34), and FGFR3G374R
heterozygotes showed achondroplasia and inhibition of bone growth
(25, 35). Thus, FGFR3 was defined in this cell type as a
negative growth regulator. Recent work demonstrated a third pathway as
shown in osteoblast cell lines at certain stages of cell
differentiation, where the signaling through FGFR3 caused apoptosis
(36). Also, in human chondrocytes expressing the mutant
FGFR3G380R, apoptosis was observed
(37). It is therefore possible that in granulosa cells
also, the activation of FGFR3 may lead to apoptosis and inhibit
granulosa function and follicular development. These signaling pathways
can be activated either by overstimulation with FGF, or by the FGFR3
mutation, which may activate the receptor in a ligand-independent
manner. However, one cannot exclude the possibility that the chronic
low levels of FSH and LH play a major role in the induction of
granulosa cell apoptosis in the FGFR3G374R
achondroplastic mice.
It was recently demonstrated that bFGF is a survival factor in
granulosa cells (15, 16, 38). In contrast, in the present
study we show that the mutant FGFR3 exerts apoptosis. This may reflect
the different pathway of FGFR signaling at different stages of cell
differentiation. For example, a recent analysis of the mutation K644E
in the kinase region of FGFR3 was shown to cause chondrocyte
proliferation on days 14 and 15 of embryonal differentiation and
inhibition of proliferation at a later stage of development on days 18
and 19 (39). Since the demonstration that FGF is a
survival factor in granulosa cells was done in cultured cells and the
apoptosis of granulosa cells shown here was during the postnatal
development of the mutant mice, this result is in agreement with the
various effects of FGFR signaling during different cell stages
(36, 37). Alternatively, the degree of activation of the
FGFR3 and its duration may determine whether it will transduce survival
or apoptotic signals (36, 37).
Significantly lower levels of FSH, LH, and progesterone in the blood of
dwarf mice, compared with normal mice (40), suggest that
the dwarf mice may have reduced amounts or lower ability to release LH
and FSH compared with normal animals. Our observation of a pronounced
reduction in bioactivity of both LH and FSH in pituitaries of dwarf
mice compared with normal animals strongly support this notion.
Alternatively, they might have a defect in releasing GnRH, which is
known to stimulate the release of LH and FSH from the pituitary to the
circulation (for review see Refs. 41 and 42).
This view is strongly supported by the pronounced effect exerted by
exogenous PMSG, which exhibits both LH/CG and FSH activity
(43), followed by the effect of hCG on rapid ovarian
development in dwarf mice, which was associated with reduction of
apoptosis and reaching sexual maturation, as was demonstrated in the
present study. This would support the notion that concerted action of
growth factors and gonadotropins is essential for ovarian development
and maturation (44, 45).
Our results on the elevated expression of p53 in the mutant ovary
suggest a possible correlation between granulosa apoptosis and p53
expression (46). Activation of p53 was previously
demonstrated to induce granulosa cell apoptosis both in
vitro and in vivo (reviewed in Ref. 47).
Nevertheless, the mechanism by which granulosa cells undergo apoptosis
in mutant mice is still obscure and requires further investigation. It
is of interest that although mutations in FGFR3 were reported mainly to
be associated with skeletal dysplasia, the receptor is expressed also
in many other tissues such as brain, lung, bladder, and ovary.
Nevertheless, no pathological effect of receptor activation was
reported in these tissues. The study reported here provides an example
of female infertility due to impairment of follicular development in
the ovary, resulting probably from activation of FGFR3 by the
achondroplastic mutation, and shows that a major effect can emerge from
a pituitary defect in the FGFR3G374R
achondroplastic mice in failing to provide optimal survival signals of
gonadotropins essential for follicular development. This effect of the
mutation was not reported for achondroplasia in humans. It is therefore
possible that it is specific to mice carrying this mutation.
Nevertheless, our data demonstrate the importance of FGFR3 in normal
development of ovarian follicles, since the mutated receptor causes a
severe defect in follicular maturation, which leads to female
infertility. Interestingly, it was recently reported that a
Ser365
Cys mutation of FGFR3 in mice led to
achondroplasia and reduced fertility of the mutant mice in which
no abnormalities were apparent in their testis and ovaries
(56). These authors suggest that the infertility may be
due to the small size of the mice. Taken together, our data and the
data of Chen et al. (56) would suggest that
different mutations in FGFR3 may lead to infertility not necessarily by
the same mechanism.
 |
MATERIALS AND METHODS
|
---|
Specific Reagents
Polyclonal antibodies to FGFR3 (C-15) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) were used both for Western
blot and immunohistochemistry. Polyclonal antibodies to mouse FGFR3
were also kindly provided by Dr. D. Ornitz (Washington University, St.
Louis, MO). Reagents for TUNEL staining were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Monoclonal
mouse Mdm2 antibodies were from Santa Cruz Biotechnology, Inc. p53-specific monoclonal antibody, mAb421 (directed against
human and mouse p53 antigen) and mAb240 (directed again mouse p53
antigen) were kindly provided by Dr. M. Oren (Weizmann Institute of
Science, Rehovot, Israel). Both goat antimouse or antirabbit IgG
coupled to horseradish peroxidase were obtained from Sigma
(Nes Ziona, Israel). A kit (Auto Prob III) for histochemical staining
of FGFR3 by the immunoperoxidase method was obtained from Biomeda Corp.
(Foster City, CA). 3H progesterone was obtained
from Sigma, Rehovot. Highly purified rat LH, FSH
[iodination grade (rat FSH-I-8, IOD; rat LH-I-9, IOD)] were obtained
from the NIH and Dr. Parlow. PMSG was obtained from N.V.
Organon (Oss, The Netherlands).
Animals
At selected time points, 3 wk- or 3-month-old normal or dwarf
mice (25) were killed by ip injection of 0.2 ml
thiopentone sodium (50 mg/ml) (Abbott S.A., Campovera, Italy),
and the ovaries, kidneys, brains, lungs, and liver were removed. One
ovary of each animal was fixed in 4% paraformaldehyde, and the other
one was snap frozen in liquid nitrogen and kept at -80 C. Liver and
other organs were also snap frozen in liquid nitrogen and kept at -80
C. The experiments were repeated three times with at least three
animals per each time point. In some experiments, 25-d-old normal and
dwarf mice were injected (nine animals of each group) with 15 IU per
animal of PMSG followed by 15 IU hCG 48 h later. Ovaries were
removed and fixed with 4% paraformaldehyde immediately after each
injection as well as 48 h after the hCG treatment.
Histology
After fixing with 4% paraformaldehyde for 24 h at 24 C,
the ovaries were embedded in 70% ethanol and subsequently immersed in
paraffin, and 6- to 10-µm sections were cut with a microtome and
stained with hematoxylin and eosin. Sections were inspected using a
Carl Zeiss light microscope (Axioscope -Carl Zeiss, Oberkochen, Germany). For examinations of apoptosis,
unstained sections were deparaffinized by washing three times with
xylene for 5 min, followed by ethanol 100%, ethanol 95%, ethanol
70%, and H2O for 3 min each. Sections were
doubly stained for terminal deoxynucleotidyl transferase-mediated nick
end labeling (TUNEL) (48) and DAPI (15, 16)
as follows: 1) TUNEL method was performed by labeling DNA strand breaks
with the In-Situ Cell Death Detection Kit (Roche Molecular Biochemicals) using fluorescein-deoxyuridine
triphosphate (dUTP). 2) After the TUNEL staining the sections were
incubated with 100 µl of 125 ng/ml of DAPI (Sigma Ltd.)
for 30 min at 24 C. Microscopic examination of the specimens was
carried out using a Carl Zeiss Axioskop microscope in a
bright field or in fluorescent mode.
Collection of Blood and Hormonal Assays
Aliquots of blood were collected from 3-month-old normal and
dwarf mice (six animals from each group). Sera were prepared at 4 C and
kept at -80 C until hormonal determinations. Pituitaries were
dissected immediately after the animals were killed and were
homogenized in 50 µl of PBS containing 1 mM of
phenylmethylsulfonyl fluoride to avoid protein degradation at 4 C.
Homogenates were kept at -80 C until hormonal determinations.
Progesterone in serum was determined by RIA as described previously
(49, 50). FSH and LH bioactivity in sera and pituitary
homogenates was determined by FSH-responsive or LH/CG-responsive cell
lines [rat FSHR-17 (see Ref. 51) and rat LHR-15
cell lines (see Ref. 52)] as described earlier
(53). In brief, the amount of progesterone secreted into
the medium in response of the appropriate cell lines to aliquots of
sera (550 µl) or 13 µl pituitary homogenates were measured, and
the bioactivities of the hormones were calculated according to
calibration curves run in parallel containing the appropriate cell
culture incubated with increasing hormone concentrations of highly
purified (iodination grade) rat LH (rat LH-I-9, IOD) or rat FSH (rat
FSH-I-8, IOD). Samples of serum and pituitary homogenates were assayed
in triplicate experiments.
Immunohistochemical Staining for FGFR3
Sections were deparaffinized, as described above, and incubated
overnight at 4 C with FGFR3 polyclonal antibodies followed by anti-IgG
coupled to peroxidase. After completing the peroxidase reaction on the
slides, the tissue was either stained or left without staining with
diluted hematoxylin solution. For nonspecific staining, sections of
microscopic slides were stained with nonimmune rabbit sera. Sections
were visualized with the Carl Zeiss microscope.
Protein Analysis and Western Blot
Frozen tissues were homogenized in lysis buffer containing: 20
mM Tris (pH 7.4), 137 mM NaCl, 2 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100,
20 µM leupeptin, 10% glycerol, 0.1% SDS, 0.5%
deoxycholate, and 5 µg/ml aprotinin. Proteins were quantified by the
Bradford method (54). Lysates containing 50 µg proteins
were boiled in sample buffer for 10 min and separated by 12% SDS-PAGE,
transferred onto nitrocellulose membranes. The blots were then blocked
using 5% milk powder in PBS plus 0.05% Tween 20 and incubated
overnight at 4 C with the corresponding first antibodies followed by
1 h incubation at 24 C with goat antirabbit or goat antimouse IgG
conjugated to horseradish peroxidase. For the detection of p53, the
first antibody solution consisted of a 1:1 mixture of mAb421 and mAb240
antibodies, which recognize different epitopes of the mouse p53 and
significantly increase the sensitivity of detection of p53 compared
with the use of each antibody alone. The detection of the second
antibody was carried out by the enhanced chemiluminescence (ECL) kit
(Amersham Pharmacia Biotech Co, Buckinghamshire, UK).
Western blot analysis was carried out in three separate
experiments.
Morphometric Analysis
Fixed ovaries embedded in paraffin were sectioned at the center
of the gland and the mean diameter was calculated to determine the size
of the ovary. Measurements were taken from six different glands that
received the same treatment. For scoring the apoptosis, at least four
sections of each ovary were used. Follicles were scored as apoptotic if
they demonstrated at least five apoptotic cells (positively stained by
the TUNEL method) in a given section. The number of corpora lutea per
gland was scored on the intact gland and confirmed by scoring the
corpora lutea in serial sections.
Oocyte Recovery and First Polar Body Extrusion
Oocytes were recovered by puncturing the antral follicles of six
ovaries of 3-month-old normal or dwarf mice and cultured for 24 h
as described recently (55). Each group (normal and dwarf
mice) contained 3040 oocytes. At the end of incubation the oocytes
were analyzed for maturation by differential interference contrast
microscopy, and the percent oocytes demonstrating extrusion of the
first polar body were scored.
Statistical Analysis
Mean values of ovarian diameter, and other morphological
measurements, and hormone values were compared using ANOVA followed by
Fishers least significance difference test. Differences
between treatment groups were considered statistically significant at
P < 0.05.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. N. Dekel from the Weizmann Institute of Science for
expertise and assistance in the analysis of the competency of oocytes
derived from normal and dwarf mice and for the photography of the
cultured oocyte through the microscope; Mr. Alon Chen from the research
team of Dr. Y. Koch at the Weizmann Institute for careful dissection of
the mice pituitaries and hypothalami; Dr. M. Oren for providing us with
antibodies to p53; and Mrs. V. Laufer for excellent secretarial
assistance.
 |
FOOTNOTES
|
---|
This work was supported by a grant (to A.A.) from the Israel Academy of
Science. A.A. is an incumbent of the Joyce and Ben B. Eisenberg
professorial chair in Molecular Endocrinology and Cancer Research.
Abbreviations: bFGF, Basic fibroblast growth factor; DAPI,
4'6'-diamido-2-phenylindole hydrochloride; FGF, fibroblast growth
factor; FGFR, FGF receptor; mAb, monoclonal antibody; TUNEL, terminal
deoxynucleotidyl transferase-mediated nick end labeling.
Received for publication November 27, 2000.
Accepted for publication June 5, 2001.
 |
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