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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). IGF-I receptor mRNA was, in contrast, present in all follicles (Fig. 1CGo). 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. 2Go). 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 (100–300 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.

 
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. 3Go). 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. 3JGo). PMSG treatment restored follicular FSHR and aromatase gene expression to normal or above-normal levels (Fig. 3Go, 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 (D–F) contains film autoradiographs of sections from a representative untreated hypophysectomized animal, and the last column (G–I) 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.

 
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. 4Go). The follicles in the IGF-I knockout ovary appear morphologically very similar to those in the FSH knockout mouse ovary (Fig. 2Go 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.

 
We examined FSHR gene expression in secondary follicles (100–300 µm in diameter) in ovaries from wild type vs. IGF-I knockout vs. IGF-I-treated knockout mice (Fig. 5Go). 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. 5Go). Note that in the wild type ovary, aromatase mRNA is expressed in follicles with the highest level IGF-I/FSHR gene expression (Fig. 5Go, A–D). 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 (A–D) 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 A–D and 100 µm for panels E–G. 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).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 FSH’s 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. 6Go).



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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.

 
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 40–100 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. 6Go). 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. 6Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 3–10 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 Mayer’s 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, 100–300 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 Fischer’s least significant difference test (Statview, Abacus Concepts, Berkeley, CA).


    ACKNOWLEDGMENTS
 
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.).


    FOOTNOTES
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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