Endocrinology Vol. 139, No. 12 5205-5214
Copyright © 1998 by The Endocrine Society
Expression of Mouse Ovarian Insulin Growth Factor System Components During Follicular Development and Atresia1
Serge-Alain Wandji,
Teresa L. Wood,
Jennifer Crawford,
Steven W. Levison and
James M. Hammond
Departments of Medicine (S.-A.W., J.C., J.M.H.), Cellular and
Molecular Physiology (J.M.H.), and Neuroscience and Anatomy (T.L.W.,
J.C., S.W.L.), Pennsylvania State University College of Medicine,
Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: James M. Hammond, Section of Endocrinology, Diabetes and Metabolism, H044, Hershey Medical Center, Hershey, Pennsylvania 17033.
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Abstract
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Insulin growth factor I (IGF-I) appears necessary for the completion of
follicular development in mice. However, little is known about changes
in the IGF system components during follicular development and
luteinization. This study determined the relation between gene
expression of specific IGF system components and follicular growth,
survival, or atresia in mice. Immature mice from three different
strains (129, C57, and MF1), with or without gonadotropin treatment
[2.5 IU PMSG/2.5 IU human CG (hCG)], were used. The strains were
similar in all parameters measured. Apoptosis, as detected by in
situ labeling of nicked DNA, preceded the appearance of
morphological signs of atresia. In healthy follicles, IGF-I transcripts
were low during the primary follicular stage but increased to a maximum
in the late preantral and early antral stages (P <
0.001) irrespective of hormone treatment. Occasionally, IGF-I
transcripts were also detected in apoptotic follicles but decreased
(P < 0.05) as a function of atresia as assessed by
morphological criteria. IGF binding protein-4 (IGFBP-4) messenger RNA
(mRNA) expression in granulosa cells was restricted to apoptotic and
atretic follicles (P < 0.001). IGFBP-5 transcript
levels, on the other hand, were elevated in granulosa cells of healthy
primary and secondary follicles but decreased in subsequent follicular
stages and in atretic follicles (P < 0.001).
Conversely, IGFBP-2 mRNA was constitutively expressed in granulosa
cells.
PMSG/hCG treatment induced the appearance of IGFBP-2 transcripts in the
ovarian interstitium. Following PMSG/hCG-induced ovulation, IGFBP-2 and
-4 and IGF type-I receptor mRNAs were strongly expressed in virtually
all luteal cells, whereas IGFBP-3 and -5 transcripts were selectively
localized to some cell types in the corpus luteum. Conversely,
IGF-I mRNA was essentially undetectable in the corpus luteum.
This study represents the most comprehensive and detailed analysis of
the physiology and anatomy of the mouse ovarian IGF system, and shows
that 1) IGFBP-5 is linked to the survival of the slow growing and
immature preantral follicles; 2) IGF-I is associated with the growth
and survival of the rapidly growing large preantral and antral
follicles; 3) IGFBP-4 is an atretogenic candidate for mouse ovarian
follicles; 4) ovulatory doses of PMSG/hCG up-regulate IGFBP-2 mRNA
expression in the ovarian interstitium; and 5) transcripts of
IGF type-I receptor and IGFBP-2 through -5, but not those of IGF-I are
highly expressed in the mouse corpus luteum.
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Introduction
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DELETION of the insulin growth factor I
(IGF-I) gene leads to failure of ovarian follicles to ovulate (1),
demonstrating that IGF-I is required for follicular development beyond
the antral stage and for completion of oocyte growth and maturation. It
has been postulated that the primary role of intraovarian IGF-I is to
amplify FSH action in vivo, because similar effects have
been demonstrated on cultured granulosa cells (1, 2, 3). All these
observations have led to the suggestion that the ultimate fate of the
follicle, i.e. growth or atresia, will depend on intrafollicular
synthesis and availability of IGF-I to interact with its cognate
receptor, the IGF type-I receptor (IGF-IR) (4). The bioavailability of
IGF-I in the ovary is regulated by several IGF binding proteins (IGFBPs
26) (5, 6, 7, 8). These components of the intraovarian IGF system have been
well documented in rodents, especially rats (6, 9, 10, 11, 12, 13, 14, 15). Considerably
less information is available about the mouse (1, 16, 17).
A more definitive characterization of the murine ovarian IGF system is
warranted for several reasons. First, the relevance of the rat
intraovarian IGF system to mouse physiology is uncertain; for instance,
IGFBP-2 messenger RNA (mRNA) is expressed in the rat
thecal-interstitial compartment, but apparently restricted to granulosa
cells in the mouse ovary (16). It has been suggested that IGF-I mRNA
expression in mouse ovaries is restricted to healthy follicles (16),
but the histological assessment of follicular health or atresia was not
documented in this study. Thirdly, to the best of our knowledge, the
existence of a correlation between specific IGFBPs and follicular
growth or atresia in the murine ovary has never been addressed.
Follicular atresia is associated with the appearance of IGFBP-4 and -5
transcripts in rat granulosa cells (12, 13, 14) and increases in IGFBP-2,
-4, and -5 in sheep antral follicles (18). In the pig, IGFBP-2 seems
most tightly correlated with atresia (19). Finally, previous studies in
the mouse were conducted in immature animals; thus, nothing is known
about gonadotropin-induced changes in the expression of IGF system
components or levels in preovulatory follicles and luteal cells. A more
extensive characterization of murine intraovarian IGFs and their
receptor and binding proteins also would provide a firm basis for
interpreting ongoing studies assessing reproductive effects of
mutations in the IGF system in mice.
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Materials and Methods
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Animals
Immature (4 weeks of age) MF1, C57, and 129 mice (purchased from
The Jackson Laboratory, Bar Harbor, ME) were killed by
cervical dislocation and their ovaries collected, immediately placed in
plastic molds containing embedding medium (Fisher Scientific International, Inc. Morris Plains, NJ), and frozen over liquid
nitrogen. In selected experiments, animals received a 2.5-IU ip
injection of PMSG (T = 0) followed by 2.5 IU of human CG (hCG)
(T = 48 h), and were killed 24 h later (T = 72
h). Ovaries were removed and immediately frozen over liquid nitrogen
vapors. Frozen ovaries were kept at -70 C until sectioned at 8 µm
thickness in a cryostat at -18 C, thaw-mounted onto Superfrost Plus
microslides (Fisher Scientific International, Inc.), air
dried, and stored at -70 C until used for in situ
hybridization or in situ end labeling of apoptotic
cells.
In situ hybridization
35S-labeled antisense complementary RNA (cRNA)
probes were synthesized from rat complementary DNA sequences for IGF-I
(20), -II (21), and -IR (22) and IGFBP-2 (23), -3 (24), -4 (25), and -5
(from Dr. Peter Rotwein, Washington University (St. Louis, MO) by
either T3 (IGFBP-3), T7 (IGF-I and -II and IGFBP-2 and -4), or Sp6
(IGFBP-5 or IGF-IR) RNA polymerase-mediated transcription of linearized
plasmids using a transcription reaction kit (Promega Corp.
Co., Madison, WI). For controls, IGF-II and IGFBP-5 sense cRNAs were
synthesized by Sp6 and T7 transcription, respectively. Unincorporated
35S-nucleotides were removed by filtration on Sephadex G50
Quick Spin columns (Boehringer-Mannheim, Indianapolis,
IN).
Sections were transferred directly from -70 C into 4%
paraformaldehyde/PBS, rinsed in PBS, dehydrated in a series of
alcohols, acetylated in 0.25% acetic anhydride (vol/vol), 0.05
M triethanolamine, 0.3% acetic acid for 10 min, washed in
0.2x SSC (1xSSC contains 0.15 M sodium chloride, 0.015
M sodium citrate), and dehydrated. Sections were
prehybridized 13 h at room temperature (RT) in 50% deionized
formamide; 0.6 M NaCl; 10 mM Tris HCl, pH 7.5;
0.02% Ficoll; 0.02% BSA; 0.02% polyvinylpyrrolidone; 1
mM EDTA; 0.1% sheared herring sperm DNA; 0.5 mg/ml yeast
total RNA; and 0.05 g/ml transfer RNA. Next, hybridization was carried
out overnight at 50 C in prehybridization buffer containing
35S-labeled cRNA probe (4 x 107 cpm/ml),
0.1 mg/ml sheared herring sperm DNA, 10% dextran sulfate, 0.1% SDS,
and 10 mM dithiothreitol. Sections were rinsed subsequently
in 2x SSC and washed in 50% formamide, 1x SSC, and 10 mM
dithiothreitol at 45 C and O.5x SSC at RT. Unhybridized probe was then
degraded by ribonuclease A [100 mg/mL in 0.5 M NaCl; 10
mM Tris, pH 7.5; and 1 mM EDTA] for 30 min at
RT, and sections were washed in 0.2x SSC at 5560 C for 2 h and
dehydrated through graded alcohols. Sections were exposed to
autoradiographic film (Kodak Biomax MR, Eastman Kodak Co., Rochester, NY) overnight to evaluate the intensity of
hybridization signals, then dipped in Kodak NTB2 liquid emulsion and
exposed for 13 weeks depending on the probe at 4 C. For a given
probe, exposure times were the same for all animals. This were 3 weeks
for IGF-I, -II, -IR, IGFBP-2, and -3 and 1 week for IGFBP-4 and -5.
Slides were developed in Kodak-D19 developer and counterstained with
Mayers hematoxylin and examined with both bright-field and dark-field
microscopy.
Quantitative analysis of in situ hybridization
Quantitative analysis of hybridization signals was performed
using a video-NIH Image Analysis program (Scion Corp., Frederick, MD)
linked to a microscope. Each probe was tested simultaneously on three
different mouse strains, and three to four animals were examined per
strain and per probe. For each animal and for each probe, one
representative section was chosen in the largest cross-section of the
ovary. Grain density was determined in a constant area of 200
µm2 using a x20 objective and dark-field illumination.
Under these conditions, there was excellent correlation between the
number of silver grains determined by visual count and image analysis.
Grain density was determined in four nonoverlapping constant (200
µm2) areas of primary or preantral follicles. Six such
areas were considered for antral follicles and corpora lutea. Only
microscopic fields completely occupied with cells were measured, except
for the background for which grain density was determined in four large
random areas (5000 µm2) devoid of tissues. For each
slide, specific labeling was determined by subtracting average
background value from the values obtained for each follicular category.
For sense probes, the specific hybridization signal determined using
these methods was negligible.
Morphological characterization of ovarian follicles
Stages of follicular development were defined as follows:
primary (a single layer of cuboidal granulosa cells), large preantral
(at least two layers of cuboidal granulosa cells and no antrum), small
antral (several layers of granulosa cells and one or several small
antral cavities but no cumulus oophorus stalk), and large antral
(several layers of granulosa cells, a single large antral cavity, and a
well-defined cumulus oophorus stalk). In preliminary studies, IGFBP-5
mRNA expression was heterogenous between subclasses of the preantral
stage. Consequently, for IGFBP-5, the large preantral stage was
further divided into secondary (two layers of granulosa cells) and
tertiary (three or more layer of granulosa cells and no antral
cavity).
Follicles were classified as healthy on the following basis: zero
(primary follicles) to no more than three pyknotic nuclei (preantral
and antral follicles), granulosa cells regularly apposed on an intact
basement membrane, no fibroblastic morphology in the granulosa cell
compartment, and no evidence of infiltration of the oocyte by somatic
cells. Follicles were classified as early atretic if they contained at
least one pyknotic nucleus in primary and four in larger follicles and
an irregular basal lamina. In addition to these criteria, late atretic
follicles also contained fibroblast cell in the granulosa cell
compartment and oocyte infiltration by somatic cells. Other criteria
for assessing follicular atresia such as dissolution of germinal
vesicle, chromatin condensation, or cytoplasmic fragmentation of the
oocyte were not easy to apply in these studies because the integrity of
these cellular compartments is not well preserved in frozen
sections.
Only follicles with a visible oocyte were considered to ensure
proper follicular counting and classification. A total of 3335
primary, 3051 healthy preantral, 1849 early atretic preantral,
640 late atretic preantral, 1226 small antral, and 012 large
antral follicles (large antral follicles were not apparent in many
sections of immature animals) were analyzed depending on the IGF system
component considered.
In situ end labeling of apoptotic nuclei
Two related but distinct procedures were used to detect
apoptosis in ovarian tissues. These procedures used either the enzyme
terminal deoxynucleotidyl transferase (TUNEL) or the Klenow fragment of
DNA polymerase I (KLUNEL) to catalyze the transfer of digoxygenin
(DIG)-labeled nucleotides to the 3'OH end of fragmented DNA. The KLUNEL
procedure has been validated in a variety of tissues (26, 27) but not
in the ovary. Conversely, the use of the TUNEL procedure to detect
apoptosis in ovaries has been previously documented (28, 29, 30). Sections
were processed identically for the two procedures except for some
differences in enzyme buffers. For the TUNEL procedure, sections were
incubated for 1 h in a buffer containing 300 U/ml Tdt
(Boehringer-Mannheim) and 0.01 M DIG DNA mixture
(Boehringer-Mannheim) in 25 mM Tris HCl, 200 mM
potassium cacodylate, and 1 mM CoCl2. For
KLUNEL, sections were incubated for 1 h at RT with 50 U/ml Klenow
polymerase I (Worthington Biochemical Corp., Lakewood, NJ)
and 0.01 M DIG DNA mixture in a buffer containing 50
mM Tris HCl, 5 mM MgCl2, 10
mM ß-mercaptoethanol, and 0.005% BSA (buffer 1).
Incubations were stopped by washing slides in either 300 mM
NaCl, 30 mM sodium citrate (TUNEL), or buffer 1 containing
0.3% Triton X-100 (KLUNEL) at RT. The remaining steps were identical
for both procedures. Sections were incubated with a 1:500 dilution of
an alkaline phosphatase-conjugated sheep anti-DIG antibody for 1 h
at 37 C, and the alkaline phosphatase reaction developed in
BCIP/NBT (Boehringer-Mannheim) for 45 sec, followed by
counterstaining with 1% methyl green.
In one series of experiments, consecutive ovarian sections from five
mice were compared sequentially with the TUNEL and KLUNEL procedures.
The same population of follicles were labeled on adjacent sections
using either DNA fragmentation assay. In a second series of
experiments, consecutive ovarian sections from five mice were used
sequentially for in situ hybridization and in
situ end labeling (KLUNEL) to determine whether or not granulosa
cells expressing IGF-I or IGFBP-2 or -4 transcripts were
apoptotic.\.
Statistical analyses
The data presented are expressed as the mean ±
SEM. Repeated measures ANOVA models were used to assess
differences between mean densities, adjusted for the average background
density, within and between follicle categories (primary through
antral, healthy, and atretic) and mouse strains for each probe (IGF-I
and IGFBP-2, -4, or -5), and for untreated and hormone-treated mouse
groups. No differences were observed between MF1, C57, and 129 for
IGF-I or IGFBP-2, -4, or -5, and consequently, results were pooled
across the strains. The covariance structure of the models accounted
for both within- and between-mouse correlation (31). Bonferroni
adjustments were made to P values and confidence intervals
for the contrasts of interest to adjust for multiple comparisons such
that the overall probability of a type I error (
) was 0.05. All
analyses were performed using the SAS statistical package (SAS Institute, Inc., Cary, NC).
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Results
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Cellular expression of components of IGF system during growth of
healthy follicles
The in situ expression of various components of the IGF
system was similar in three different mouse strains (129, C57, and
MF1). IGF-I mRNA expression was restricted to the granulosa cell
compartment of a set of follicles in untreated (Fig. 1
) and hormone-treated immature mice
(Fig. 2A
). In morphologically healthy
follicles, IGF-I mRNA levels were lower (P < 0.001) in
primary follicles than in larger preantral and antral follicles in
untreated (Fig. 3
) and hormone-treated
immature mice (not shown). In granulosa cells of healthy antral
follicles, IGF-I mRNA was expressed as a gradient; the signal was
stronger in the cumulus oophorus and in the vicinity of the antrum than
in the mural compartment (P < 0.001; Fig. 3
).
Irrespective of hormonal treatment, IGF-II mRNA signal was mainly
expressed in ovarian blood vessels; in addition, there was minor
diffuse signal in the rest of the ovary (data not shown).

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Figure 1. In situ localization of message for
IGF system components in ovaries of untreated immature mice. Dark-field
micrographs of representative mouse ovarian sections hybridized with
IGF-I and -IR and IGFBP-2, -3, -4, and -5 cRNA probes. IGF-I and -IR
and IGFBP-2 transcripts are localized to granulosa cell compartment.
IGFBP-3 mRNA is expressed in a few cells within thecal compartment.
IGFBP-4 mRNA is constitutively expressed in thecal/interstitial
compartment and selectively expressed in granulosa cells of a few
follicles. IGFBP-5 mRNA is localized to ovarian cortex and to granulosa
cells of small preantral follicles with less than three layers of
granulosa cells.
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Figure 2. In situ localization of message for
IGF system components in ovaries of PMSG/hCG-treated mice. A,
Dark-field micrographs of representative mouse ovarian sections
hybridized with IGF-I and -IR and IGFBP-2, -3, -4, and -5 cRNA probes.
Immature (4 weeks old) mice received an ip injection of 2.5 IU PMSG
followed by 2.5 IU hCG 48 h later and were killed 24 h after
hCG. Location of various signals is same as in untreated animals. In
addition, gonadotropin treatment induced IGFBP-2 mRNA expression in
ovarian interstitium. Transcripts of IGF-IR and IGFBP-2, -3, -4, and -5
but not IGF-I are all present in corpus luteum (cl). B, Dark-field
micrographs of IGFBP-3 mRNA expression in a representative mouse
ovarian section at high magnification. Upper panel, A strong
hybridization signal is present in a few cells in periphery of some
follicles and in ovarian interstitium. Area identified by
arrow was further magnified in lower panel. Lower
panel, High magnification Normanski interference contrast view of
follicular walls. Follicular IGFBP-3 mRNA expression is restricted to a
few cells within thecal compartment (TC). GC, granulosa cell
compartment. Bars, 50 µm.
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Figure 3. Quantitative distribution of IGF-I mRNA as a
function of stage of follicular development in untreated immature mice.
Ovarian sections of untreated immature mice were hybridized with
35S-labeled antisense probe as described in
Materials and Methods. Amount of mRNA was expressed as
number of grains occupying a constant area (grain density) within
granulosa cell compartment. Cum, Cumulus/antral granulosa cells;
Mural, mural granulosa cells. Bars without common
superscript letters are different (P
< 0.001).
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In untreated (Fig. 1
) and gonadotropin-treated immature mice (Fig. 2A
),
IGFBP-2 mRNA was detected in the granulosa cell compartment of most
follicles from the primary to antral stage. IGFBP-2 transcripts were
also present in some primordial follicles (not shown). In healthy
follicles, the level of IGFBP-2 transcripts did not change as a
function of follicular development (Fig. 4
). A strong induction of IGFBP-2 mRNA
expression was also observed in the ovarian interstitium of
PMSG/hCG-treated (Fig. 2A
) but not of untreated immature mice (Fig. 1
).

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Figure 4. Quantitative distribution of IGFBP-2 and -4 as a
function of stage of follicular development in untreated immature mice.
Ovarian sections of untreated immature mice were hybridized with
35S-labeled antisense probes as described in
Materials and Methods. Amount of mRNA was expressed as
number of grains occupying a constant area (grain density) within
granulosa cell compartment. No significant differences were observed.
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IGF-IR mRNA was constitutively expressed in the granulosa cell
compartment of most follicles from all stages, from primary to antral,
healthy or atretic (Figs. 1
and 2A
). IGFBP-3 mRNA was strongly
expressed in some interstitial cells and isolated cellspresumably
endothelialin the thecal compartment of some large preantral and
antral follicles in untreated (Fig. 1
) and hormone-treated mice (Fig. 2
, A and B). Modest and relatively constant levels of IGFBP-3
transcripts were also detected in peripheral follicular cells at all
stages of follicular development (Fig. 2B
). IGFBP-4 transcripts were
constitutively expressed in the thecal and interstitial compartments in
ovaries of untreated (Fig. 1
) and hormone-treated mice (Fig. 2A
).
However, in granulosa cells of healthy follicles, IGFBP-4 transcripts
were very low throughout follicular development (Figs. 1
, 2A
, and 4
).
Irrespective of hormone treatment, IGFBP-5 transcript levels were
highest in the germinal epithelium of the ovary and in granulosa cells
of healthy follicles during the primary and secondary stages but
decreased sharply (P < 0.001) in late preantral and
subsequent stages (Figs. 1
, 2A
, and 5
). A
closer examination revealed IGFBP-5 mRNA expression as early as the
primordial follicular stage (not shown).

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Figure 5. Quantitative distribution of IGFBP-5 mRNA as a
function of stage of follicular development in untreated immature mice.
Ovarian sections of untreated immature mice were hybridized with
35S-labeled antisense probe as described in
Materials and Methods. Amount of mRNA was expressed as
number of grains occupying a constant area (grain density) within
granulosa cell compartment. Bars without common
superscript letters are different (P
< 0.001).
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In summary, the localization of expression for most IGF system
components was relatively constant during follicle growth. Changes in
the location of expression was noted for IGFBP-2, which appeared in the
interstitium after hormonal treatment. Changes in intensity of
expression were most notable for IGF-I, which increased with follicle
growth and IGFBP-5 which showed a biphasic profile.
IGF-I and IGFBP-2, -4, and -5 mRNA expression in relation to
follicular atresia
The levels of IGF-I and IGFBP-2, -4, and -5 mRNA were determined
quantitatively as a function of histological criteria for follicular
health or atresia. There was an inverse relationship between the levels
of IGF-I mRNA expression and the stage of atresia in preantral
follicles of untreated (P < 0.05; Fig. 6
) or gonadotropin-treated immature
(P < 0.01; results not shown) mice. There was no
difference in the level of IGFBP-2 mRNA expression between healthy and
early atretic follicles during the preantral stage, but there was a
decrease in IGFBP-2 expression in late atretic stages
(P < 0.05; Fig. 6
). In granulosa cells, IGFBP-4
transcripts were very low in healthy preantral (Fig. 6
) and antral
(results not shown) follicles but increased proportionally with the
stage of atresia (P < 0.001, Fig. 6
). These results
are in contrast with constitutive expression in the theca noted above.
IGFBP-5 mRNA expression was highest in healthy primary and secondary
follicles and decreased with each stage of atresia (P
< 0.001, Fig. 7
).

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Figure 6. Changes in IGF-I and IGFBP-2 and -4 mRNA
distribution in healthy and atretic preantral follicles of untreated
immature mice. Ovarian sections of untreated immature mice were
hybridized with 35S-labeled antisense probes as described
in Materials and Methods. Follicular health or atresia
was assessed by histological criteria. Amount of mRNA for each probe
was expressed as number of grains occupying a constant area (grain
density) within granulosal compartment. Within each IGF system
component, bars without common superscript
letters are different (IGF-I, P < 0.05;
IGFBP-2, P < 0.05; IGFBP-4, P
< 0.001).
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Figure 7. Changes in IGFBP-5 mRNA distribution as a function
of health or atresia of mouse preantral follicles. Ovarian sections of
untreated immature mice were hybridized with 35S-labeled
IGFBP-5 antisense probe as described in Materials and
Methods. Follicular health or atresia was assessed by
histological criteria. Amount of mRNA for each probe was expressed as
number of grains occupying a constant area (grain density) within
granulosal compartment. Bars without common
superscript letters are different (P
< 0.001).
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IGF-I and IGFBP-2 and -4 mRNA expression in relation to apoptosis
of granulosa cells
Programmed cell death is the mechanism leading to initiation and
progression of follicle cell atresia (32, 33). Therefore, it was
further hypothesized that DNA fragmentation would be linked to a
decrease in IGF-I mRNA or an increase in IGFBP-2 or -4 transcripts in
follicle cells. In preliminary studies, two DNA fragmentation assays
(TUNEL) and (KLUNEL) tested on consecutive sections produced similar
results (Fig. 8
). The presence of
apoptotic cells clearly preceded morphological signs of atresia. On the
other hand, follicles showing morphological signs of atresia also
contained apoptotic granulosa cells, except for some late stage atretic
follicles that were completely depleted of granulosa cells. When
consecutive sections were used sequentially for in situ
hybridization (IGF-I or IGFBP-2 or -4 mRNA) and in situ end
labeling (KLUNEL) of fragmented DNA, IGF-I mRNA was always expressed in
healthy follicles with at least two layers of granulosa cells, but
occasionally, a positive signal was also detected in apoptotic
follicles (Figs. 9
, A and B). Transcripts
of IGFBP-2 mRNA were detected in both apoptotic and nonapoptotic
follicles (Figs. 9
, C and D). In contrast, granulosa cell expression of
IGFBP-4 mRNA was restricted to apoptotic follicles (Figs. 9
, E and
F).

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Figure 8. Comparison between in situ end
labeling of apoptotic nuclei using either Tdt (TUNEL) or Klenow
fragment of DNA polymerase (KLUNEL). Consecutive murine ovarian
sections were processed for either TUNEL (A) or KLUNEL (B) procedure to
detect apoptotic cells as described in Materials and
Methods. Essentially the same population of follicles was
labeled (arrows) with either procedure.
Bar, 150 µm.
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Figure 9. Relation between expression of IGF-I and IGFBP-2
and -4 mRNA and apoptosis of granulosa cells. Consecutive mouse ovarian
sections were processed for in situ hybridization or
in situ end labeling of apoptotic cells as described in
Materials and Methods. A, Dark-field illumination of an
ovarian section hybridized with IGF-I cRNA probe. B, Consecutive
section subjected to in situ end labeling (KLUNEL) of
apoptotic nuclei. Healthy growing follicles (arrows)
always strongly hybridize to 35S-labeled IGF-I probe,
whereas follicles containing apoptotic cells had either a weak
(single arrowhead) or strong (double
arrowheads) IGF-I mRNA signals. C and D, Consecutive ovarian
sections subjected to in situ hybridization of IGFBP-2
mRNA and in situ end labeling, respectively. Both
healthy (arrows) and atretic follicles
(arrowheads) strongly express IGFBP-2 mRNA. E and F,
Consecutive ovarian sections subjected to in situ
hybridization of IGFBP-4 mRNA and in situ end labeling,
respectively. Granulosa cell IGFBP-4 mRNA expression is low in healthy
follicles (arrows) but strong in follicles containing
apoptotic cells (arrowheads).
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mRNA expression of components of IGF-I system in corpus luteum of
PMSG/hCG-treated mice
IGF-I (Fig. 10A
) and -II mRNA (not
shown) were low or absent in the corpus luteum, whereas transcripts of
IGF-IR and IGFBP-2 were easily detected in virtually all luteal cells
(Fig. 10
, B and C, respectively). In contrast to the absence of IGFBP-4
mRNA expression in granulosa cells of healthy follicles, its transcript
levels increased rapidly in luteinizing cells and were present in some
luteal cells following gonadotropin treatment (Fig. 10E
). As mentioned
above, IGFBP-3 mRNA expression during follicular development was
restricted to a few cells of the thecal compartment in antral
follicles. Following ovulation, some cells morphologically identical to
those IGFBP-3-positive cells appeared to infiltrate the forming corpus
luteum (Fig. 10D
). Other luteal cell types were negative for IGFBP-3
mRNA expression (Fig. 10D
). Similarly, IGFBP-5 transcripts were
expressed in some but not all luteal cells (Fig. 10F
).

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Figure 10. Gene expression of IGF system components in mouse
corpus luteum. Dark-field micrographs of IGF-I (A), IGF-IR (B), IGFBP-2
(C), IGFBP-3 (D), IGFBP-4 (E), and IGFBP-5 (F) hybridization to mouse
luteal cells. There was little IGF-I mRNA expression in luteal cells
(A). Note strong diffuse expression of IGF-IR (B) and IGFBP-2 (C).
IGFBP-4 (E) was more heterogeneous but present in both granulosa- and
theca-derived lutein cells. In contrast, IGFBP-3 (D) and IGFBP-5 (F)
transcripts are restricted to a few discrete luteal cells. Sections
were lightly counterstained with Harris hematoxylin.
Bar, 150 µm.
|
|
The cellular localization of transcripts of mouse ovarian IGF system
components is summarized in Table 1
.
View this table:
[in this window]
[in a new window]
|
Table 1. Gene expression of IGF system components in various
ovarian compartments of unstimulated and hormone-treated immature mice
|
|
 |
Discussion
|
|---|
The current work establishes that transcripts of IGF-I, -II, -IR
and IGFBP-2 through -5 are expressed in the mouse ovary. However, only
the expression of IGF-I and IGFBP-4 and -5 (but not IGFBP-2) mRNA
critically change during the course of follicular development. Previous
studies have indicated the expression of many of these IGF system
components in murine ovaries (16, 17) but have not provided a complete
picture of changes during follicular growth or atresia.
In the current study, the involvement of IGF-I in follicular
development is suggested by a rapid increase of its transcript levels
as follicle growth progresses toward the antral stage. IGF-I was also
linked to follicular survival in the current study, because the levels
of IGF-I mRNA were inversely correlated with atresia as determined
morphologically. It has been shown that apoptotic cell death with
accompanying DNA fragmentation is the molecular mechanism underlying
the initiation and progression of follicular atresia (32). Our results
show that early apoptosis was not always associated with a decrease in
IGF-I transcript levels. In fact a decrease in IGF-I mRNA was only
detected once morphological signs of atresia had developed. Therefore,
a decrease in IGF-I mRNA levels is likely a consequence, rather than a
cause of granulosa cell apoptosis. It has been previously reported that
follicular development becomes arrested at the early antral stage in
IGF-I null mutant mice (1). That study (1) and another (17) indicate
that IGF-I regulates antrum formation and FSH action (increased
aromatase activity) in granulosa cells of antral follicles.
Collectively, these observations clearly demonstrate a requirement for
IGF-I in follicular development beyond the antral stage. However, IGF-I
is not essential before antrum formation because preantral follicles
can develop in IGF-I knockout mice (1). Nonetheless, the presence of
IGF-IR transcripts in granulosa cells of preantral follicles suggests
earlier development of IGF responsivity. It is noteworthy that early
stages of follicular development, e.g. primary follicles,
which are characterized by a low growth rate (34, 35), were associated
with low IGF-I expression in our study, whereas IGF-I increased to a
maximum in rapidly growing large preantral and early antral follicles
(34, 35). Together, these observations suggest that IGF-I may be rate
limiting for preantral follicle development.
In contrast to the studies with IGF-I, few previous studies link the
IGFBPs to follicular development or death in the mouse. Adashi et
al. (1997) noted that IGFBP-2 was expressed predominantly in the
granulosa compartment in agreement with our current studies, a
situation different than in the rat (16). However, these authors failed
to note a strong positive or negative correlation between IGFBP
expression and follicle health or atresia. In contrast, numerous data
have linked IGFBP-2 (19), -4 (13, 14), and -5 (12, 14) with atresia in
other species. Surprisingly, granulosa cell IGFBP-2 mRNA expression,
which was abundant, failed to correlate with any measure of follicular
development or atresia in the current study.
Our current observations that IGFBP-4 mRNA is expressed in granulosa
cells of some but not all follicles is consistent with a previous
report (16). In our study, we also determined that IGFBP-4 mRNA
expression in granulosa cells was restricted to the same follicles in
which apoptotic granulosa cells were detected. Consistent with these
results, follicles showing histological signs of atresia also expressed
IGFBP-4 transcripts in their granulosa cell compartment. Interestingly,
the expression of IGFBP-4 transcripts in granulosa cells preceded the
appearance of morphological signs of atresia. Together, these data
suggest that IGFBP-4 could be a marker of incipient follicular atresia
in the mouse ovary. It is possible that IGFBP-4 promotes follicular
atresia by sequestrating IGF-I, thereby reducing its availability to
interact with IGF-IR in granulosa cells.
Transcripts of IGFBP-5, like those of IGF-I, were inversely correlated
with follicular atresia. In contrast, it has been reported that in the
rat ovary, IGFBP-5 mRNA is restricted to atretic preantral follicles
(12, 14). The confinement of IGFBP-5 transcripts to granulosa cells
of healthy primary and secondary follicles is remarkable because these
follicles have the slowest growth rate (35) and a limited steroidogenic
capacity (36). The capability to synthesize estradiol is acquired by
granulosa cells later, during the antral stage, and IGF-I regulates
gonadotropin stimulation of aromatase activity (36, 17). Therefore it
is possible that IGFBP-5 prevents the premature differentiation, i.e.
steroidogenic capability, of granulosa cells in healthy primary and
secondary follicles, perhaps by sequestrating IGF-I. Alternatively, the
recent identification of IGFBP-5 receptors in other systems raises the
possibility of an IGF-independent action of this binding protein in
preantral follicles (37, 38, 39).
Our observations suggest that the mouse corpus luteum is a source of
all the binding proteins considered (IGFBP-2 through -5) but not of
IGF-I or -II. The absence of in situ transcripts of the IGFs
and the expression of IGF-IR and IGFBP-2 to -5 to luteal cells suggest
that extraovarian IGF-I or -II traveling through the ovarian
microvasculature could supply the corpus luteum. In fact, recent
studies in humans suggest that extraovarian IGF-IGFBP complexes may
reach the ovarian follicular fluid as well (40). Very little is known
about the role of IGFBPs in the regulation of luteal function. It is
possible that IGFBP-3 and -5, both of which can form a ternary complex
with the IGFs and the acid-labile subunit (41), target circulating IGF
to luteal cells. IGFBP-4 has generally manifested an inhibitory action
on the ovary (42), and in our study, atresia and luteinization were
both marked by an increase in IGFBP-4 mRNA and a decrease in IGF-I
transcripts in luteal and granulosa cells. Granulosa cells of atretic
and luteinizing follicles also produce more progesterone and less
estradiol (36). Collectively, it is possible that IGFBP-4 regulates
granulosa cell luteinization, perhaps by sequestrating IGF-I, therefore
reducing its availability for IGF-IR. Similarly, in the pig ovary,
IGFBP-4 mRNA expression has been reported to increase in preovulatory
follicles and to be associated with granulosa cell luteinization
(43).
Although the actions of the IGFBPs in the ovary are incompletely
understood, the data from multiple studies suggests that the effects of
IGFBP-4 are the most consistently inhibitory (see Ref. 44 for a
review). In contrast, IGFBP-2, -3, and -5 have each been shown to
exhibit both positive and negative effects on cellular function in a
variety of culture systems (44, 45). Although stimulatory effects of
these IGFBPs have not yet been demonstrated in ovarian cells, the
correlation of expression of some proteins, e.g. IGFBP-5, with
follicular growth and development suggests that this may be plausible
in vivo.
In summary, we demonstrated that critical changes occur in the level of
expression of IGF-I and IGFBP-4 and -5 genes during follicular growth
and atresia in mice. Changes in IGF-I transcripts parallel the ability
of preantral follicles to move from a slow to a rapid growth phase.
During this period of slow growth, IGFBP-5 is strongly expressed,
suggesting that it may regulate IGF effects. A major finding of this
study is that IGFBP-4 is an atretogenic candidate for mouse ovarian
follicles; the appearance of its transcripts in granulosa cells
coincides with the hallmark of apoptosis (DNA fragmentation) and
precedes morphological signs of follicular atresia.
 |
Acknowledgments
|
|---|
We express our thanks to Mr. Allen R. Kunselman, Biostatistics
Section, Department of Health Evaluation Sciences for assistance in
statistical analyses, and Dr. Kang Li for preparation of histological
sections.
 |
Footnotes
|
|---|
1 This work was supported by NIH Grant HD-24565 (to J.M.H.) and an NIH
fellowship (to S.A.W). 
Received June 29, 1998.
 |
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