Insulin-Like Growth Factor I Regulates Gonadotropin Responsiveness in the Murine Ovary
Jian Zhou,
T. Rajendra Kumar,
Martin M. Matzuk and
Carolyn Bondy
Developmental Endocrinology Branch (J.Z., C.B.) National
Institute of Child Health and Human Development National Institutes
of Health Bethesda, Maryland 20892
Department of
Pathology (T.R.K.), Molecular and Human Genetics and Cell Biology
(M.M.M.) Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT
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The present study shows that insulin-like growth
factor I (IGF-I) and FSH receptor (FSHR) mRNAs are selectively
coexpressed in a subset of healthy-appearing follicles in murine
ovaries, irrespective of cycle stage. Aromatase gene expression, a
prime marker for FSH effect, is found only in IGF-I/FSHR- positive
follicles, showing that these are healthy, gonadotropin-responsive
follicles. Given the striking coexpression of FSHR and IGF-I, we
hypothesized that FSH was responsible for follicular IGF-I expression.
We found, however, that granulosa cell IGF-I mRNA levels are not
reduced in hypophysectomized (±PMSG) or FSH knockout mice, indicating
that FSH does not have a major role in regulation of granulosa cell
IGF-I gene expression. To test the alternative hypothesis that IGF-I
regulates FSHR gene expression, we studied ovaries from IGF-I knockout
mice. FSHR mRNA was significantly reduced in ovaries from homozygous
IGF-I knockout compared with wild type mice and was restored to control
values by exogenous IGF-I treatment. The functional significance of the
reduced FSHR gene expression in IGF-I knockout ovaries is suggested by
reduced aromatase expression and by the failure of their follicles to
develop normally beyond the early antral stage. In fact, IGF-I knockout
and FSH knockout ovaries appear very similar in terms of arrested
follicular development. In summary, we have shown that IGF-I and FSHR
are selectively coexpressed in healthy, growing murine follicles and
that FSH does not affect IGF-I expression but that IGF-I augments
granulosa cell FSHR expression. These data suggest that ovarian
IGF-I expression serves to enhance granulosa cell FSH responsiveness by
augmenting FSHR expression.
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INTRODUCTION
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Ovarian follicular growth begins and proceeds to the preantral
stage autonomously, i.e. independently of gonadotropin
regulation (1). Further development depends upon FSH acting upon its
cognate receptor expressed by granulosa cells (2). FSH receptor (FSHR)
activation results in elevation of granulosa cell cAMP, activation of
A-kinase, phosphorylation of cAMP-response element-binding protein, and
consequent alterations in cAMP-response element-regulated gene
expression (3, 4). At present it is not known whether these FSH-induced
signals directly enhance granulosa cell proliferation and aromatase
expression, or whether they instigate the production of local mediators
such as insulin-like growth factor I (IGF-I), which then stimulate
proliferation and estrogen synthesis.
Previous studies have shown that IGF-I is selectively expressed in a
subset of relatively healthy-appearing follicles in the rat ovary (5, 6), leading to the suggestion that IGF-I is a marker for follicular
selection. More recently, we demonstrated a highly significant positive
correlation between granulosa cell IGF-I gene expression and DNA
synthesis in murine ovaries (7). Since IGF-I enhances the proliferation
of many cell types and since we have previously shown that the IGF-I
receptor is coexpressed with IGF-I in ovarian follicles (6), it seemed
likely that IGF-I may act in an autocrine/paracrine manner to stimulate
granulosa cell proliferation. The mechanism by which IGF-I is
selectively induced in a subset of candidate dominant follicles is
unknown. Given the considerations mentioned above, we hypothesized that
FSH may stimulate follicle growth by inducing granulosa cell IGF-I
production.
In the present study we have attempted to elucidate the
functional relationship between FSH and IGF-I in ovarian follicle
growth. If FSH regulates follicular IGF-I synthesis, then the FSHR
should be expressed in the same subset of follicles that express IGF-I.
Thus, we compared IGF-I and FSHR gene expression in serial ovarian
sections. Upon finding a pattern of selective coexpression for IGF-I
and FSHR mRNAs, we then asked whether FSH regulates IGF-I expression or
whether IGF-I regulates FSHR expression, comparing their responses to
hypophysectomy and gonadotropin treatment, and expression in FSH and
IGF-I knockout mice.
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RESULTS
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To elucidate the relationship between FSH action and IGF-I
expression in the murine ovary, we compared FSHR and IGF-I gene
expression in serial ovarian sections from random cycling, lactating,
and pregnant rats. In all ovaries, FSHR mRNA was detected only in IGF-I
mRNA-positive follicles (Fig. 1
). IGF-I
receptor mRNA was, in contrast, present in all follicles (Fig. 1C
).
Identical findings were obtained in mice (Ref. 8 and see below). To
determine whether FSH induces granulosa cell IGF-I gene expression,
IGF-I gene expression was evaluated in FSH knockout mice (Fig. 2
). IGF-I mRNA demonstrates the same
selective pattern of distribution and the same abundance in granulosa
cells from FSH knockout mice ovaries as in wild type controls.

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Figure 1. IGF-I and FSHR Coexpression in the Mature Rat Ovary
Panel A is an hematoxylin and eosin-stained section, and panels B-D are
film autoradiographs of sequential sections through the same ovary,
hybridized to RNA probes for IGF-I (B), IGF-I receptor (C), and FSHR
(D). IGF-I receptor mRNA characterizes all follicles, while a select
subset of follicles demonstrate IGF-I and FSHR mRNAs.
Arrowheads indicate follicles that are IGF-I/FSHR
negative. This ovary was obtained from a pregnant rat; identical
results were found in lactating and cycling rats and mice irrespective
of cycle stage. cl, Corpus luteum. Bar = 1 mm.
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Figure 2. IGF-I Gene Expression in Wild Type (A and B) and
FSH Knockout (C and D) Mouse Ovaries
Four FSH knockout ovaries were hybridized to the IGF-I cRNA probe, and
the paired bright and darkfield micrographs shown here are
representative of the results. No significant difference in the level
of IGF-I mRNA was detected in size-matched (100300 µm diameter)
follicles from FSH knockout (n = 4) and wild type (n = 4)
ovaries, P = 0.497. WT, Wild type; FSH KO, FSH
knockout. Bar = 100 µm.
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To further examine the role of FSH in ovarian IGF-I and FSHR
expression, we evaluated IGF-I, FSHR, and aromatase gene expression
after hypophysectomy, in the presence or absence of gonadotropin (PMSG)
replacement. Aromatase mRNA was used as a functional marker for FSH
effects. Hypophysectomy in the presence or absence of gonadotropin
treatment had no effect on the level of IGF-I mRNA expression (Fig. 3
). FSHR mRNA, however, was reduced by
approximately 40%, and aromatase mRNA was reduced by more than 90%
after hypophysectomy in the absence of gonadotropin replacement (Fig. 3J
). PMSG treatment restored follicular FSHR and aromatase gene
expression to normal or above-normal levels (Fig. 3
, G-I). These data
indicate that FSH does not regulate IGF-I gene expression but does
significantly augment FSHR gene expression as well as aromatase gene
expression in IGF-I-positive granulosa cells.

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Figure 3. Effects of Hypophysectomy on IGF-I (A, D, and G),
FSHR (B, E, and H) and Aromatase (C, F, and I) Gene Expression in the
Murine Ovary
The first column (A-C) contains film autoradiographs of sequential
ovarian sections from a representative sham-operated animal. The middle
column (DF) contains film autoradiographs of sections from a
representative untreated hypophysectomized animal, and the last column
(GI) has autoradiographs from a representative
hypophysectomized/PMSG-treated animal. These data are from newly
studied right ovaries of animals from a study reported in Ref. 6 in
which the left ovaries were used to evaluate IGF-I and IGF-I receptor
mRNA levels. ft, Fallopian tube. Bar = 0.6 mm.
Panel J, Graphic analysis of changes in IGF-I, FSHR, and aromatase
(ARO) mRNAs in the different groups. Data were obtained by computerized
image analysis as described in Materials and Methods and
is expressed as means ± SEM. There were four animals
each in the sham and hypophysectomy groups and two in the PMSG group.
The P values are in comparison with sham-operated.
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To examine an alternative explanation for IGF-I and FSHR coexpression,
i.e. the possibility that IGF-I regulates FSHR gene
expression, we investigated FSHR and aromatase mRNAs in ovaries from
IGF-I knockout mice. IGF-I knockout females are infertile and do not
appear to undergo puberty (8). Their ovaries are proportionate to their
body size and have a normal complement of oocytes and primordial
follicles (8) but demonstrate arrested follicular development at the
preantral/early antral stage with no mature graafian or luteinized
follicles being detected (Fig. 4
). The
follicles in the IGF-I knockout ovary appear morphologically very
similar to those in the FSH knockout mouse ovary (Fig. 2
and Refs. 1
and 8). There was no increase in the number of atretic follicles (Ref.
8 and the present study) and no apparent increase in granulosa cell
programmed death (our unpublished data) in IGF-I knockout ovaries.

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Figure 4. Morphology of the IGF-I Knockout Ovary
These ovaries are from 3-month-old wild type (WT, panels A and B) and
homozygous knockout (KO, panels C and D) littermates. Panels C and D
show follicular morphology at a higher magnification. cl, Corpus
luteum; ca, corpus albicans; g, graafian follicle.
Bar = 200 µm for panels A and C and 50 µm for
panels B and D.
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We examined FSHR gene expression in secondary follicles (100300
µm in diameter) in ovaries from wild type vs. IGF-I
knockout vs. IGF-I-treated knockout mice (Fig. 5
). FSHR mRNA was reduced by
approximately 50% (P = 0.0015) in IGF-I knockout
granulosa cells but was restored to wild type levels after 2 weeks of
exogenous IGF-I treatment (P = 0.017, Fig. 5
). Note
that in the wild type ovary, aromatase mRNA is expressed in follicles
with the highest level IGF-I/FSHR gene expression (Fig. 5
, AD).
Aromatase mRNA levels were also dramatically reduced in the IGF-I
knockout mice and appeared increased in the IGF-I-treated knockout
mice, but they were not quantified because too few follicles expressed
aromatase.

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Figure 5. Normal Patterns of FSHR, Aromatase, and IGF-I Gene
Expression in Serial Sections from a Wild Type Ovary (AD) Compared
with FSHR Gene Expression in IGF-I Knockout/Saline-treated (F) and
IGF-I Knockout/IGF-I-Treated (G) Mice
Bar = 200 µm for panels AD and 100 µm for
panels EG. Panel H demonstrates quantification of FSHR mRNA levels
graphically. Wild type, n = 13; IGF-I knockout, n = 8; IGF-I
knockout/IGF-I-treated, n = 3. FSHR mRNA levels were reduced by
approximately 50% (P = 0.0015) in IGF-I knockout
granulosa cells but were restored to wild-type levels after 2 weeks of
exogenous IGF-I treatment (P = 0.017).
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DISCUSSION
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The present study has shown that granulosa cell IGF-I and FSHR
gene expression are selectively colocalized in a subset of healthy
growing and ultimately selected follicles in the murine ovary. Further,
we have shown that FSH does not regulate granulosa cell IGF-I gene
expression, since the latter appears normal in FSH knockout ovaries and
is unperturbed by hypophysectomy in normal animals. Conversely, the
fact that granulosa cell FSHR gene expression is significantly reduced
in IGF-I knockout mice demonstrates that IGF-I normally augments FSHR
expression. The reduction in FSHR mRNA in IGF-I knockout follicles
appears to be of major functional significance, since aromatase
expression is also reduced and graafian follicle development is
defective in IGF-I knockout ovaries. Thus it appears that a critical
level of FSHR expression, normally ensured by local IGF-I action,
is essential for gonadotropin responsiveness and follicular
de-velopment.
Further support for the view that IGF-I regulates FSHR expression comes
from in vitro studies on murine granulosa cells in which the
major effect of IGF-I is to augment FSHs actions. For example, IGF-I
amplifies FSH-induced aromatase expression and LH receptor induction
(9, 10). We have recently shown that the porcine ovary also
demonstrates selective follicular IGF-I and FSHR coexpression (11) and
interestingly, IGF-I also amplifies FSH effects on porcine granulosa
cells in vitro (12). This suggests that IGF-I acts primarily
by augmenting FSHR expression in both murine and porcine follicles. In
contrast, IGF-II rather than IGF-I is expressed by human granulosa
cells (13, 14, 15), and IGF-II expression is not linked to FSHR expression
in the primate ovary (our unpublished data). IGF-I and IGF-II have a
variety of in vitro actions on human granulosa cells that
are not FSH-dependent (reviewed in Ref.16). Thus it seems that
follicular IGF-I and FSH coexpression is indicative of a functional
relationship in which IGF-I increases FSHR expression and thus
potentiates FSH action.
Our previous study demonstrated a highly significant correlation
between local IGF-I expression and granulosa cell DNA synthesis and
suggested that IGF-I might directly stimulate granulosa cell
proliferation (6). However, follicles in IGF-I knockout ovaries appear
to have a normal complement of granulosa cells, at least up to the late
preantral or early antral stage (8). Thus, if IGF-I has a role in
granulosa cell proliferation, it must be during the late, FSH-dependent
granulosa proliferation occurring from early antral to preovulatory
follicle development. It has been suggested that local IGF-I serves to
prevent granulosa cell-programmed death (17). However, granulosa cell
apoptosis and follicular atresia do not appear to be increased in IGF-I
knockout ovaries (8). It is possible that IGF-I is protective of
granulosa cell survival under in vitro conditions (17), but
that in vivo other trophic factors serve this purpose.
While we have shown that IGF-I significantly augments FSHR gene
expression in murine granulosa cells in vivo, we have not
established whether this is a direct or indirect effect of IGF-I
action. Regulation of FSHR gene expression is poorly understood at
present, and it is possible that IGF-I has a primary effect on
granulosa cell maturation that results secondarily in augmentation of
FSHR gene expression. IGF-I is not essential for induction of granulosa
cell FSHR gene expression de novo, since FSHR mRNA is still
present, albeit at low levels, in the IGF-I knockout ovary. It is
possible, however, that this FSHR mRNA is not efficiently translated in
the absence of IGF-I, since IGF-I knockout mice do not respond to
gonadotropin treatment for ovulation induction (8). There is evidence
for a dissociation between the presence of FSHR transcripts and
FSH-responsiveness, possibly due to the expression of alternatively
spliced mRNAs encoding a nonfunctional receptor (reviewed in Ref.18).
A persistent question concerning the process of ovarian follicular
selection is why only a few follicles develop in response to
gonadotropins when all are exposed to equal circulating levels. The
present study indicates that exposure to FSH effect is not
equal among follicles, since selective IGF-I expression significantly
amplifies FSHR gene expression and presumably FSH action in a subset of
follicles. Amplification by IGF-I of FSHR expression is positively
reinforced by FSH-induced augmentation of IGF-I receptor expression,
which has been demonstrated in vivo at the mRNA level (6)
and in vitro at the IGF-I-binding level (19). Thus, local
IGF-I expression creates an intrafollicular positive feedback loop in
which IGF-I enhances FSH action and FSH enhances IGF-I action through
mutual complementary receptor up-regulation (Fig. 6
).

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Figure 6. Schematic Illustrating Proposed Role for IGF-I in
Follicular Development
Oocyte autonomous signals are hypothesized to initiate granulosa cell
proliferation and IGF-I production. IGF-I augments FSHR expression, and
FSH augments both IGFIR and FSHR expression. This mutual complementary
postive feedback loop within the follicle is hypothesized to be
critical for the amplification of FSH action to induce the formation of
mature graafian follicles. In the absence of IGF-I or FSH, follicles
arrest at a preantral/early antral stage of development. Maximal FSH
action leads to mature antrum formation and granulosa cell aromatase
and LHR expression. The resulting peak in follicular E2
synthesis stimulates an LH surge, which in turn stimulates ovulation.
For the sake of simplicity, thecal layer development, which is also
impaired in the IGF-I knockout mouse, has been omitted from the
diagram.
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Although the present study provides evidence that local IGF-I
expression is responsible for selective follicular responsiveness to
FSH, it has not addressed the issue of how selective follicular IGF-I
expression comes about. GH treatment increases ovarian IGF-I levels
(20), but in the absence of GH (e.g. after hypophysectomy),
granulosa IGF-I mRNA remains abundant and selectively expressed (6, 21), suggesting that GH is not responsible for regulating normal IGF-I
expression in the ovary. Since granulosa cell IGF-I production begins
after the onset of oocyte growth and stops shortly after granulosa
cells are put into culture [and hence separated from the oocyte,
(22)], it seems likely that oocyte-derived signals stimulate granulosa
cell IGF-I synthesis. We first suggested this view based upon the
observation that granulosa IGF-I mRNA is most abundant in cells closest
to the oocyte (6). For example, it is possible that a soluble factor
such as GDF-9 secreted by oocytes (23) triggers and sustains granulosa
cell IGF-I production. If this hypothesis is true, the selectivity in
follicular IGF-I expression reflects the vigor of the cohort of
activated oocytes.
The reduced level of granulosa cell FSHR expression in IGF-I knockout
ovaries may explain the infertility of the IGF-I knockout females (8).
Follicles in the IGF-I knockout ovary are arrested at a late preantral
or early antral stage of development. A graafian follicle reported
in the previous study (8) may have represented a large secondary
follicle with degenerating luminal granulosa cells, as has been
reported in FSH-deficient mice (1). Mature graafian follicles with well
developed antrums were not observed in the present analysis of ovaries
from six additional animals, aged 40100 days. FSH is necessary for
normal antrum formation (1), and the absence of antralization in the
IGF-I knockout ovary is hypothesized to be due to diminished FSHR
expression and thus inadequate FSH effects (Fig. 6
). The follicles in
IGF-I knockout ovaries are also immature with respect to thecal
development. The largest follicles in IGF-I knockout ovaries have a
thecal layer that consists of thin layers of fibroblast-like cells
without discernible internal and external layers and without the
epithelioid cells indicative of exuberant steroidogenesis. The
explanation for this observation may be that granulosa cell-derived
IGF-I has a paracrine role in thecal development. Alternatively, other
factors produced by granulosa cells in response to FSH may normally
stimulate thecal development and may be deficient in the IGF-I knockout
ovary due to inadequate FSH effect. The former hypothesis seems more
likely in view of the fact that thecal development appears relatively
normal in the FSH knockout mouse ovary (1).
In any case, due to inadequate follicular aromatase expression and
inadequate thecal development, it is predicted that IGF-I knockout mice
fail to produce the normal midcycle rise in estradiol necessary for the
LH surge and thus do not ovulate spontaneously (Fig. 6
). These mice
also fail to ovulate in response to exogenous gonadotropins (8),
probably because FSH is necessary for normal granulosa cell LH receptor
expression (2, 18). Thus, we hypothesize that IGF-I absence causes
primary gonadal failure due to gonadotropin resistance at the level of
the granulosa cell, and that ovarian IGF-I normally serves to entrain
murine follicular development to gonadotropin regulation.
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MATERIALS AND METHODS
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Animals
Ovaries were obtained from random cycling, pregnant, and
lactating rats of the Sprague-Dawley strain and mice of the MF-1 and
CD1 strains (Taconic Farms, Germantown, NY). Ovaries from homozygous
IGF-I knockout mice (8, 24, 25), homozygous FSH knockout (1), and wild
type littermates were obtained at 40 and 100 days of age. One group of
IGF-I knockout mice (n = 3) received treatment with recombinant
human IGF-I (10 µg/g, Genentech, South San Francisco, CA) given
intraperitoneally twice a day for 2 weeks from P28 to P41. All ovaries
were snap frozen and stored at -70 C. Frozen sections of 10 µm
thickness were cut at -15 C, thaw-mounted onto
poly-L-lysine-coated slides and stored at -70 C until
hybridization. All animals were used in accord with protocols approved
by the NICHD Animal Care and Use Committee.
Hypophysectomy
Female Sprague-Dawley rats were hypophysectomized (hx) or
sham-operated at 20 days of age at Taconic Farms and shipped to us 5
days after the procedure. All animals were given free access to food
and water containing 5% sucrose. The hypophysectomy and hormone
treatment protocol has been described in detail (6). Four hx rats
received 50 IU PMSG ip (Gestyl, Organon, West Orange, NJ), four hx rats
received a saline injection, and the four sham-operated rats also
received a saline injection.
RNA Probes
The structure and synthesis of the 35S-labeled cRNA
probes for IGF-I and the IGF-I receptor have been described in detail
previously (6). A sense probe was synthesized from the IGF-I receptor
template. The FSHR cDNA (26) was a gift from Ares Advanced Technology
(Randolph, MA). This 2118-bp fragment was subcloned into pSV.Sport
(Lise Technology, Rockville, Md). Sense and antisense templates were
linearized with EcoRI and KpnI, respectively. A
273-bp fragment (27) encoding aromatase was generated by PCR.
The sequence of the 3'-oligo-nucleotide was 5'- TTGTTGTTAAATATGATGCC-3'
and that of the 5'-oligonucleotide was 5'-ATACCAGGTCCTGGCTACTG-3'. PCR
was carried out on 1 ng of human placenta cDNA in 100-µl reactions
with 1 µM primer, 200 µM deoxynucleoside
triphosphates, 1x Taq buffer, and 2.5 U of Taq
polymerase (Perkin-Elmer, Norwalk, CT) using a cycling program of 93 C
for 2 min followed by 93 C for 1 min, 40 C for 1 min, and 72 C for 1
min for 30 cycles. The PCR amplification product was then ligated into
pCR II vector (Invitrogen, San Diego, CA), and the orientation was
determined by DNA sequencing (Applied Biosystems, Foster City, CA).
In Situ Hybridization
Before hybridization, sections were warmed to 25 C, fixed in 4%
formaldehyde, and soaked for 10 min in 0.25% acetic anhydride/0.1
M triethanolamine hydrochloride/0.9% NaCl. Tissue was then
dehydrated through an ethanol series, delipidated in chloroform,
rehydrated, and air-dried. The 35S-labeled probes
(107 dpm/ml or approximately 50 ng/ml) were added to
hybridization buffer composed of 50% formamide, 0.3 M
NaCl, 20 mM Tris-HCl, pH 8, 5 mM EDTA, 500 µg
transfer RNA/ml, 10% dextran sulfate, 10 mM
dithiothreitol, and 0.02% each of BSA, Ficoll, and
polyvinylpyrolidone. After the 35S-labeled probe in
hybridization buffer was added to the sections, coverslips were placed
over the sections, and the slides were incubated in humidified chambers
overnight (14 h) at 55 C.
Slides were washed several times in 4 x standard sodium citrate
to remove coverslips and hybridization buffer, dehydrated, and immersed
in 0.3 M NaCl, 50% formamide, 20 mM Tris-HCl,
1 mM EDTA at 60 C for 15 min. Sections were then treated
with ribonuclease A (20 µg/ml) for 30 min at room temperature,
followed by a 15-min wash in 0.1 x standard sodium citrate at 50
C. Slides were air-dryed and apposed to Hyperfilm-beta Max (Amersham,
Arlington Heights, IL) for 310 days and then dipped in Kodak NTB2
nuclear emul-sion (Eastman Kodak, Rochester, NY), stored with
desiccant at 4 C for 15 (IGF-I) or 30 days (FSHR, IGF-I receptor sense
and antisense, and aromatase), developed, and stained with Mayers
hematoxylin and eosin for microscopic evaluation.
Quantification of mRNA
IGF-I, FSHR, and aromatase mRNAs were quantified by image
analysis using darkfield illumination on a Leitz DM RX microscope
connected to a Macintosh PowerPC-based computer analysis system. Hybrid
signal was measured over granulosa cells in secondary follicles,
100300 µm in diameter, measured from basement membrane (not
including the theca) across the largest diameter of the follicle.
Grains overlying an area of 500 µm2 were captured at
400x via a solid state monochrome video camera and the data were
analyzed using the NIH Image v1.57 software. Background signal obtained
from ovarian connective tissue in each section was subtracted from
totals for the same section before further analysis. Data on mRNA
levels were compared using ANOVA, and differences between means were
evaluated by Fischers least significant difference test (Statview,
Abacus Concepts, Berkeley, CA).
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ACKNOWLEDGMENTS
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We are indebted to Argiros Efstratiadis (Columbia University,
New York, NY) and Lynn Powell-Braxton (Genentech, San Francisco, CA)
for providing their respective IGF-I knockout mice lines for these
experiments. We also thank Ricardo Dreyfuss for expert
photomicrography.
These studies were supported in part by NIH Grant CA-60651 (to
M.M.M.).
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FOOTNOTES
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Address requests for reprints to: Jian Zhou, MD, PhD, Building 10, Room 10N262, NIH, Bethesda, Maryland 20892.
Received for publication February 13, 1997.
Revision received September 12, 1997.
Accepted for publication September 15, 1997.
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REFERENCES
|
---|
-
Kumar TR, Wang Y, Lu N, Matzuk MM 1997 FSH is required
for ovarian follicle maturation but not male fertility. Nat Genet 15:201204[Medline]
-
Uilenbroek JT; Richards JS 1979 Ovarian follicular
development during the rat estrous cycle: gonadotropin receptors and
follicular responsiveness. Biol Reprod 20:11591165[Medline]
-
Adashi EY, Resnick CE, Jastorff B 1990 Blockade of granulosa
cell differentiation by an antagonistic analog of adenosine
3',5'-cyclic monophosphate (cAMP): central but non-exclusive
intermediary role of cAMP in follicle-stimulating hormone action. Mol
Cell Endocrinol 7:111
-
Habener JF, Miller CP, Vallejo M 1995 cAMP-dependent
regulation of gene transcription by cAMP response element-binding
protein and cAMP response element modulator. Vitam Horm 51:157[Medline]
-
Oliver JE, Aitman JR, Powell JF, Wilson CA, Clayton RN 1989 IGF-I gene expression in the rat ovary is confined to the granulosa
cells of developing follicles. Endocrinology 124:26712679[Abstract]
-
Zhou J, Chin E, Bondy CA 1991 Cellular pattern of IGF-I and
IGF-I receptor gene expression in the developing and mature follicle.
Endocrinology 129:32813288[Abstract]
-
Zhou J, ReFeuzo J, Bondy CA 1995 Granulosa cell DNA synthesis
is strictly correlated with the presence of IGF-I and absence of AP-1
expression in vivo. Mol Endocrinol 9:924932[Abstract]
-
Baker J, Hardy MP, Zhou J, Bondy CA, Lupu F, Bellve AR,
Efstratiadis A 1996 Effects of an IgfI gene null mutation on mouse
reproduction. Mol Endocrinol 10:903918[Abstract]
-
Adashi EY, Resnick CE, Brodie AM, Svoboda ME, Van Wyk JJ 1985 Somatomedin-C-mediated potentiation of follicle-stimulating
hormone-induced aromatase activity of cultured rat granulosa cells.
Endocrinology 117:23132320[Abstract]
-
Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ 1985 Somatomedin-C enhances induction of luteinizing hormone receptors by
follicle-stimulating hormone in cultured rat granulosa cells.
Endocrinology 116:23692375[Abstract]
-
Zhou J, Adesanya OO, Vatzias G, Hammond JM, Bondy CA 1996 Selective expression of insulin-like growth factor system components
during porcine ovary follicular selection. Endocrinology 137:48934901[Abstract]
-
Hammond JM, Mondschein JS, Samaras SE, Canning SF The ovarian
insulin-like growth factors, a local amplification mechanism for
steroidogenesis hormone action. J Steroid Biochem Mol Biol 40:411416
-
Hernandez ER, Hurwitz A, Vera A, Pellicer A, Adashi EY,
LeRoith D, Roberts Jr CT 1991 Expression of the genes encoding the
insulin-like growth factors and their receptors in the human ovary.
J Clin Endocrinol Metab 74:419425[Abstract]
-
Zhou J, Bondy CA 1993 Anatomy of the human ovarian IGF system.
Biol Reprod 48:467482[Abstract]
-
el-Roeiy A, Chen X, Roberts VJ, Shimasakai S, Ling N, LeRoith
D, Roberts Jr CT, Yen SS 1994 Expression of the genes encoding the
insulin-like growth factors (IGF-I and II), the IGF and insulin
receptors, and IGF-binding proteins-16 and the localization of their
gene products in normal and polycystic ovary syndrome ovaries. J
Clin Endocrinol Metab 78:14881496[Abstract]
-
Giudice LC 1992 Insulin-like growth factors and ovarian
follicular development. Endocr Rev 13:641669[Medline]
-
Chun SY, Billig H, Tilly JL, Furuta I, Tsafriri A, Hsueh AJ 1994 Gonadotropin suppression of apoptosis in cultured preovulatory
follicles: mediatory role of endogenous insulin-like growth factor I.
Endocrinology 135:18451853[Abstract]
-
Richards JS 1993 Gonadotropin regulated gene expression in the
ovary. In: Adashis EY, Leung PCK (eds) The Ovary. Raven Press, New
York, pp 93111
-
Adashi EY, Resnick CE, Hernandez ER, Svoboda ME, Van Wyk JJ 1988 In vivo regulation of granulosa cell
somatomedin-C/IGF-I receptors. Endocrinology 122:13831389[Abstract]
-
Davoren JB, Hsueh AJ 1986 Growth hormone increases ovarian
levels of immunoreactive somatomedin C/insulin-like growth factor I
in vivo. Endocrinology 118:888890[Abstract]
-
Hernandez ER, Roberts Jr CT, LeRoith D, Adashi EY 1989 Rat
ovarian insulin-like growth factor I (IGF-I) gene expression is
granulosa cell-selective: 5'-untranslated mRNA variant representation
and hormonal regulation. Endocrinology 125:572574[Abstract]
-
Botero LF, Roberts CT, LeRoith D, Adashi EY, Hernandez ER 1993 IGF-I gene expression by primary cultures of ovarian cells: insulin and
dexamethasone dependence. Endocrinology 132:27032708[Abstract]
-
Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM 1996 Growth differentiation factor-9 is required during early
ovarian folliculogenesis. Nature 383:531535[CrossRef][Medline]
-
Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis 1993 Mice carrying null mutations of the genes encoding insulin-like growth
factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:5972[Medline]
-
Powell-Braxton L, Hollingshead P, Warburton C, Dowd M,
Pitts-Meek S, Dalton D, Gillett N, Stewart TA 1993 IGF-I is required
for normal embryonic growth in mice. Genes Dev 7:26092617[Abstract]
-
Kelton CA, Cheng SV, Nugent NP, Schweickhardt RL, Rosenthal
JL, Overton SA, Wands GD, Kuzeja JB, Luchette CA, Chappel SC 1992 The
cloning of the human follicle stimulating hormone receptor and its
expression in COS-7, CHO, and Y-1 cells. Mol Cell Endocrinol 89:141151[CrossRef][Medline]
-
Price T, Aitken J, Head J, Mahendroo M, Means G, Simpson E 1992 Determination of aromatase cytochrome P450 messenger ribonucleic
acid in human breast tissue by competitive polymerase chain
amplification. J Clin Endocrinol Metab 74:12471252[Abstract]