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Endocrinology Vol. 138, No. 9 3881-3890
Copyright © 1997 by The Endocrine Society

ARTICLES

The Mouse Intraovarian Insulin-Like Growth Factor I System: Departures from the Rat Paradigm1

Eli Y. Adashi, Carol E. Resnick, Donna W. Payne, Ron G. Rosenfeld, Tomoko Matsumoto, Maya K. Hunter, Sharron E. Gargosky, Jian Zhou and Carolyn A. Bondy

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Maryland School of Medicine (E.Y.A., C.E.R., D.W.P.), Baltimore, Maryland 21201; the Department of Pediatrics, University of Oregon Health Sciences Center (R.G.R., T.M., M.K.H., S.E.G.), Portland, Oregon 97201; the Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (J.Z., C.A.B.), Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Department of Obstetrics and Gynecology, University of Utah Health Science Center, Suite 2B 200, 50 North Medical Drive, Salt Lake City, Utah 84132. E-mail: eadashi{at}Hsc.utah.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the rat intraovarian insulin-like growth factor I (IGF-I) system is well documented, the increasing availability of null mouse mutants for components of the IGF system necessitates characterization of the mouse model as well. Therefore, we undertook to define the components of the mouse intraovarian IGF-I system and to examine its operational characteristics. The cellular pattern of ovarian gene expression was comparable in the immature rat and mouse for IGF-I and the type I IGF receptor. In both species, IGF-I messenger RNA (mRNA) is selectively expressed by granulosa cells in growing, healthy appearing follicles. Type I IGF receptor mRNA was also concentrated in granulosa cells, but was uniformly expressed in all follicles large and small, healthy and atretic appearing alike. Cellular patterns of IGF-binding protein (IGFBP) gene expression were similar in mouse and rat, except in the case of IGFBP-2. IGFBP-2 mRNA was localized to the mouse granulosa cell, in contrast to its concentration in the rat thecal-interstitial compartment. This difference in IGFBP expression pattern was also noted in cultured mouse and rat granulosa cells. Although immunoreactive IGFBP-4 (24 and 28 kDa) and IGFBP-5 (29 kDa) were shared by both species, the cultured mouse granulosa cell also featured immunoreactive IGFBP-2 (30 kDa). The mouse paradigm further differed from its rat counterpart in that a maximal dose of FSH, previously shown to suppress the elaboration of rat granulosa cell-derived IGFBPs, was without effect. The addition of IGF-I proved stimulatory to the accumulation of the 28- to 29-kDa IGFBPs, as previously reported for the rat. However, IGF-I proved inhibitory to the accumulation of the 24-kDa IGFBP (presumptive nonglycosylated IGFBP-4); no consistent effect was reported for the rat model. Functional comparisons of mouse and rat ovarian cell cultures revealed qualitatively comparable FSH-stimulated steroidogenesis, disposition of radiolabeled pregnenolone, IGF-I-amplified FSH action, and IGFBP-mediated antigonadotropic activity. These findings indicate that the mouse intrafollicular IGF-I system differs from the rat paradigm in both the makeup and regulation of granulosa cell-derived IGFBPs as well as in the intensity and character of the steroidogenic process. Studies employing the mouse model must take into account these important distinctions relative to the more established rat paradigm.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A GROWING BODY of information relevant to the rat paradigm (1–26) has documented the existence of a complete intraovarian insulin-like growth factor I (IGF-I) system, replete with a ligand (IGF-I) (1, 2, 3, 4, 5, 6, 7, 8, 9), a receptor (type I) (9, 10, 11, 12, 13, 14, 15, 16), and IGF-binding proteins (IGFBP-2 to -6) (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27). According to current views, the primary if not sole role of bioavailable intrafollicular IGF-I may be the amplification of FSH action in granulosa cells (28, 29). It is this FSH-amplifying property of IGF-I that underlies the hypothesis that the intrafollicular IGF-I system may be a determinant of follicular fate (30). More specifically, it has been hypothesized that net intrafollicular IGF-I activity may effect follicular selection by way of FSH amplification, thereby distinguishing follicles destined to ovulate from those destined to succumb to atresia (30).

Although the intraovarian IGF system has been carefully delineated in several species (31, 32), relatively little attention has been paid to the mouse model. Indeed, most rodent work has been limited to the rat (1–27), an established experimental model in the context of ovarian physiology. Recently, the relative significance of the mouse paradigm has substantially increased because of the availability of null mouse mutants generated by targeted gene disruption. At the time of this writing, the selective obliteration of mouse IGF-I (29, 33, 34, 35), IGF-II (36, 37), type I IGF receptor (33, 34), IGFBP-2 (38), or combinations thereof (33, 34) have been reported. Undoubtedly, additional components of the IGF system are currently the subject of targeted elimination.

In light of the above, we set out to characterize the intraovarian IGF-I system of the MF1 mouse, a strain used in several of the null mutants established to date (29, 33, 34). Specifically, we have defined the components of the mouse intraovarian IGF-I system and compared it with its rat counterpart. Moreover, we examined the operational characteristics of isolated granulosa cell cultures and whole ovarian dispersates of mouse origin. Special emphasis has been placed on FSH responsiveness, the ability of IGF-I to amplify FSH action, and the ability of IGFBPs to exert an antigonadotropic effect. Additional efforts were directed at the miniaturizing of mouse culture systems given projected limitations in the number of animals and/or ovarian cells available for study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Immature MF1 female mice (25–32 days old), purchased from Harlan Sprague-Dawley (Indianapolis, IN), were killed by CO2 asphyxiation. The choice of the MF1 strain was dictated by its recent use in the generation of several null mutant mice relevant to the IGF system (29, 33, 34).

Materials
McCoy’s 5{alpha} medium (modified, without serum), penicillin-streptomycin solution, L-glutamine, trypsin/EDTA, BSA, deoxyribonuclease, and trypan blue stain (0.4%) were obtained from Life Technologies (Gaithersburg, MD). Collagenase (Clostridium histolyticum; CLS type I; 144 U/ml) was purchased from Worthington Biochemical Corp. (Freehold, NJ). [7-N-3H]pregnenolone (10.5 Ci/mmol) was obtained from DuPont-New England Nuclear Research Products (Boston, MA). Androstenedione was obtained from Sigma Chemical Co. (St. Louis, MO).

Ovine FSH (oFSH; NIH FSH S18; FSH potency equal to 65.6 NIH FSH S1 units/mg) was a gift from the National Hormone and Pituitary Program (Rockville, MD) through the NIDDK, NICHHD, and USDA. Recombinant human (h) IGF-I was purchased from Bachem (Torrance, CA). Synthetic IGF-II was provided by Dr. C. H. Li, University of California-San Francisco. Recombinant hIGFBP-2 was provided by Dr. Jean-Luc Mary, University of Basel (Basel, Switzerland). Recombinant IGFBP-3 was provided by Dr. Christopher A. Maack, Biogrowth (Richmond, CA).

Tissue culture procedures
Isolated granulosa cells were obtained by repeated follicular puncture as previously described for the rat model (7). Cell viability, as assessed by trypan blue dye exclusion, was consistently 40% or more.

Whole ovarian dispersates were prepared and cultured as previously described for the rat model (39). Briefly, ovaries were dissected free, washed (twice) with 5 ml McCoy’s 5a medium (serum-free), divided into four to six pieces, and subjected to enzymatic dispersion for 60–90 min at 37 C using 0.4% (wt/vol) collagenase, 0.001% (wt/vol) deoxyribonuclease, and 0.1% (wt/vol) BSA. In the course of this incubation period, the ovaries were dissociated into a cell suspension by repeated pipetting every 30 min with a graded series of micropipettes (id, 0.5–1.0 mm). At the end of the dispersion period, the cells were collected by centrifugation at 250 x g for 5 min, washed (three times) with McCoy’s 5a medium, and then resuspended into a known volume of the same. Cell viability, as assessed by trypan blue dye exclusion, was consistently 74% or more.

All cells were inoculated onto 16-mm, 24-well tissue culture plates (Costar, Cambridge, MA) containing 0.5 ml McCoy’s 5a medium (serum-free) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin sulfate (100 µg/ml) and maintained for up to 72 h at 37 C under a water-saturated atmosphere of 95% air and 5% CO2. All agents were dissolved in sterile culture medium and applied in 50-µl aliquots. At the conclusion of the incubation period, the media were collected before further processing as described below.

Western ligand blotting
Synthetic IGF-II was iodinated by a modification of the chloramine-T technique to specific activities of up to 250 µCi/µg. The iodinated peptide was then purified by gel filtration over a Sephadex G-50 column (1.0 x 120 cm) at 4 C and eluted with 100 mM HEPES buffer, pH 7.4, containing 0.5% BSA, 120 mM NaCl, 1.2 mM MgSO4, 5 mM KCl, 50 mM sodium acetate, and 10 mM dextrose.

Conditioned media or serum were electrophoresed on SDS-PAGE (10%) under nonreducing conditions. The size-fractionated proteins were then electroblotted onto nitrocellulose for 1 h. Thereafter, the filter-immobilized proteins were blocked, incubated overnight at 4 C with 1 x 106 cpm [125I]IGF-II, washed, and visualized by autoradiography according to the method of Hossenlopp et al. (40). Mol wt were estimated using prestained protein standards. Densitometric analysis of autoradiograms was performed on an Ultrascan XL laser densitometer (LKB, Stockholm, Sweden).

Immunoprecipitation
Immunoprecipitation of IGFBPs was performed by directly adding 2–10 µl/sample of 1:5 diluted hIGFBP-2-directed antibody ({alpha}-Hec1A) antiserum (41), hIGFBP-4-directed antibody (Austral Biologicals, San Ramon, CA), hIGFBP-5-directed antibody (Austral Biologicals), or normal rabbit serum (nonimmune control). The antibodies in question have never been fully validated in the murine system, but are assumed to identify the appropriate IGFBPs. After an overnight incubation at 4 C, precipitation was achieved by the addition of 50 µl Pansorbin (Calbiochem-Novabiochem International, La Jolla, CA), followed by centrifugation. The precipitates were then washed three times, boiled for 5 min in SDS buffer, and subjected to Western ligand blotting as previously described (40).

Complementary DNAs
The rat IGF-I probe contained 376 bases complementary to part of the IGF-I-coding region as well as to 3'-untranslated region sequences (42). The rat type I IGF receptor probe contained 265 bases complementary to 15 bases of 5'-untranslated region, the region encoding the signal peptide, and the first 53 amino acids of the {alpha}-subunit (43). The rat IGFBP-2 clone consisted of a 585-bp fragment corresponding to nucleotides 502-1087 (44). The rat IGFBP-3 clone represented a 440-bp coding region EcoRI-BamHI fragment (45). The hIGFBP-4 probe was synthesized off a 462-bp complementary DNA corresponding to the midportion of the protein-coding region (46). The mouse IGFBP-5 clone, generously provided by Dr. Peter Rotwein, Washington University (St. Louis, MO), constituted a 463-bp fragment encoding the first 150 amino acids of the mature protein. Sense control probes were synthesized from the IGF-I and IGF-I receptor inserts and hybridized in parallel experiments.

In situ hybridization
Freshly obtained ovaries were flash-frozen, sectioned at -15 C at 10-µm thickness, thaw-mounted onto polylysine-coated slides, and stored at -70 C until use. 35S-Labeled RNA probes were synthesized to a specific activity of 2 x 108 dpm/µg in a protocol that has been previously described and validated in detail (47). 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, and 0.95% NaCl. Tissue was then dehydrated through an ethanol series, dilapidated in chloroform, rehydrated, and air-dried. The 35S-labeled probes (107 dpm/ml or ~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 polyvinylpyrrolidone. 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 5 x SSC (standard saline citrate) to remove coverslips and hybridization buffer, dehydrated, and immersed in 0.3 M NaCl, 50% formamide, 20 mM Tris-HCl, and 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 SSC at 50 C. Slides were air-dried, apposed to Hyperfilm-Beta Max (Amersham, Arlington Heights, IL) for 3–7 days, then dipped in Kodak NTB2 nuclear emulsion (Eastman Kodak, Rochester, NY), stored with desiccant at 4 C for 15 days, developed, and stained with Mayer’s hematoxylin and eosin for microscopic evaluation. The signal from sense control probes hybridized, exposed, and developed in parallel experiments was minimal.

RIAs
The medium progesterone content was determined using a specific antiserum (no. 337) raised against progesterone-11-BSA (48) provided by Dr. Gordon D. Niswender, Colorada State University (Ft. Collins, CO). Assay procedures, performance characteristics, and specificity were previously described (49).

The total medium estrogen content was determined using an antiserum raised against estradiol-17ß-O-carboxymethyloxime bovine thyroglobulin (50) provided by Dr. Delwood C. Collins (formerly of Emory University School of Medicine, Decatur, GA). Assay procedures, performance characteristics, and specificity were previously described (49).

HPLC
Medium steroids were extracted (twice) with ethyl acetate (3 ml), and the steroid-containing solvent layer was evaporated to dryness. The steroids were then dissolved in hexane-isopropyl alcohol (95:5) and separated on a silica gel column containing a chemically bonded diol phase (10 µm; LiChrosorbDiol, EM Reagents, Gibbstown, NJ) with the use of a Waters (Milford, MA) HPLC system as previously described (51).

Data analysis
Data are presented as the mean ± SE of multiple experiments (n noted in figure legends), each with replicate assays. RIA data analysis was carried out using Curve Fit software (developed by Munson, D. Rodbard, and M. L. Jaffe, NIH, Bethesda, MD), a package based on the four-parameter logistic equation designed to fit the results to a sigmoidal function curve. Statistical significance was determined by ANOVA (Fisher’s protected least significant differences test) or Student’s t test, as noted. Statistics were calculated using StatView 512+ for Macintosh (Brain Power, Calabasas, CA) and Instat for Macintosh (GraphPad Software, InStat for MacIntosh, San Diego, CA). Statistical significance (P < 0.05) is denoted by asterisks in the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mouse intraovarian IGF-I system: in situ localization of system components
The object of this study was to determine which ovarian cell types synthesize IGF-I, the type I IGF receptor, and IGFBP-2 to -6. It was not designed to quantitatively compare expression of specific RNAs in mouse vs. rat. In situ detection of IGF-I, type I IGF receptor, and IGFBP-1 to -6 was performed on ovaries from six 25-day-old MF1 mice. Results were consistent in all six animals. Representative serial sections from the ovary of one of these mice are displayed in Fig. 1Go. Type I IGF receptor messenger RNA (mRNA; Fig. 1BGo) was concentrated in granulosa cells and was uniformly expressed in all follicles, large and small, healthy and atretic appearing alike. IGF-I mRNA (Fig. 1CGo) was detected in granulosa cells of medium sized, healthy appearing follicles, with the signal most prominent in antral or cumulus granulosa. Lesser abundance was noted for mural granulosa cells or larger follicles. IGFBP-1 mRNA was not detected (not shown). IGFBP-2 mRNA (Fig. 1DGo) was detected in granulosa cells of all follicles. IGFBP-3 mRNA was detected in some ovarian blood vessels, but not in follicles (not shown). IGFBP-4 mRNA (Fig. 1EGo) was detected primarily in the thecal-interstitial and stromal compartments. Occasional granulosa cells did, however, display a signal for IGFBP-4. IGFBP-5 mRNA (Fig. 1FGo) was concentrated in the granulosa cells of small follicles located in the cortical region of the ovary. IGFBP-5 mRNA was also heavily concentrated in the germinal epithelium, but was barely detected (i.e. in a nondescript pattern) in the stromal compartment. IGFBP-6 mRNA proved very scarce (data not shown).



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  		Figure 1. The mouse intraovarian IGF-I system: pattern of gene expression in the immature 25-day-old mouse. A, Brightfield micrograph of a hematoxylin- and eosin-stained ovarian section; B–F, darkfield micrographs of serial ovarian sections hybridized to RNA probes for the type I IGF receptor (IGFR; B), IGF-I (C), IGFBP-2 (D), IGFBP-4 (E), and IGFBP-5 (F). Bar = 200 µm.

 
The present findings in the mouse were compared with previous data for the rat at comparable stages of development, and the comparative data are summarized in Table 1Go. All IGF system components demonstrate apparently identical patterns of ovarian gene expression for the two species with the notable exception of IGFBP-2. Indeed, whereas IGFBP-2 localized to the mouse granulosa cell (Fig. 1DGo), it localized exclusively to the thecal-interstitial compartment in the immature (Fig. 2Go) as well as the mature rat (18).


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  		Table 1. Cellular gene expression patterns the mouse vs. rat intraovarian IGF-I system

 


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  		Figure 2. IGFBP-2 mRNA in the immature 30-day-old rat ovary. The paired brightfield (A) and darkfield (B) micrographs reveal IGFBP-2 mRNA to localize to the thecal-interstitial (ti) compartment of the rat ovary. No expression was detected in granulosa cells. Bar = 200 µm.

 
Mouse granulosa cell-derived IGFBPs: effects of treatment with FSH or IGF-I
To further establish the pattern of granulosa cell-derived IGFBPs, isolated mouse granulosa cells (1 x 105 viable cells/dish) were cultured for 48 h under serum-free conditions in the absence of treatment or in the presence of FSH (4 mIU/ml) or FSH plus IGF-I (100 ng/ml). At the conclusion of the experimental period, media were collected and subjected to Western ligand blotting. As shown (Fig. 3Go), untreated granulosa cells elaborated a modest ligand-blottable 28- to 29-kDa doublet along with a major 24-kDa species. Importantly, treatment with a maximal stimulatory dose of FSH was without effect on the relative accumulation of the IGFBPs. In contrast, provision of a maximal stimulatory dose of IGF-I up-regulated the accumulation of the 28- to 29-kDa doublet; the reverse phenomenon was noted for the 24-kDa IGFBP species. Qualitatively comparable results were obtained when granulosa cells were exposed to IGF-I alone (Fig. 4Go).



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  		Figure 3. Mouse granulosa cell-derived IGFBPs: effect of treatment with FSH or IGF-I. Granulosa cells (1 x 105 viable cells/dish) were cultured for 72 h under serum-free conditions in the absence of treatment or in the presence of FSH (2 mIU/ml) or FSH plus IGF-I (100 ng/ml). Thereafter, media were collected and subjected to Western ligand blotting as described in Materials and Methods. Data reflect a representative gel. Qualitatively comparable results were obtained in three additional experiments.

 


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  		Figure 4. Mouse granulosa cell-derived IGFBPs: effect of treatment with IGF-I. Granulosa cells (1 x 105 viable cells/dish) were cultured for 72 h under serum-free conditions in the absence or presence of IGF-I (100 ng/ml). Thereafter, media were collected and subjected to Western ligand blotting as described in Materials and Methods. Data reflect a representative gel. Qualitatively comparable results were obtained in one additional experiment. NHS, Normal human serum.

 
To better establish the identity of the IGFBP bands, media conditioned by untreated or IGF-I (100 ng/ml)-treated granulosa cells were subjected to immunoprecipitation with antibodies directed against IGFBP-2, -4, and -5, as described in Materials and Methods. As shown (Fig. 5Go), immunoprecipitation with an IGFBP-2-directed antibody revealed a 30-kDa band not readily apparent when assessed by Western ligand blotting. Immunoprecipitation with an IGFBP-4-directed antibody revealed a major 24-kDa band and a relatively minor 28-kDa band. Immunoprecipitation with an IGFBP-5-directed antibody revealed a 29-kDa band. Although incubation with nonimmune rabbit serum appeared to be associated with a yet to be defined ligand-blottable IGFBP, the band in question displayed a molecular mass distinct from that noted for IGFBP-2, and -5. Western ligand blotting of normal human serum served as a positive control, depicting the well described array of circulating human IGFBPs. Taken together, these findings suggest that the 24-kDa species is comprised of immunoreactive IGFBP-4, whereas the 28- to 29-kDa is comprised of both immunoreactive IGFBP-4 and IGFBP-5. In addition, a 30-kDa band compatible with immunoreactive IGFBP-2 was seen. Treatment with IGF-I appeared to increase the relative representation of the IGFBP-5 protein while decreasing its IGFBP-4 counterpart (Figs. 3Go and 4Go).



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  		Figure 5. Mouse granulosa cell-derived IGFBPs: immunoprecipitation studies. Media conditioned by untreated or IGF-I (100 ng/ml)-treated granulosa cells were subjected to immunoprecipitation with antibodies directed against IGFBP-2, -4, and -5, as described in Materials and Methods. Media were also subjected to incubation with no antibody or with nonimmune/normal rabbit serum. The resultant precipitates were subjected to Western ligand blotting as described in Materials and Methods. Data reflect a representative gel. Qualitatively comparable results were obtained in one additional experiment.

 
Steroidogenic characteristics of the mouse intraovarian IGF-I system: cultured granulosa cells
To assess the size of the cellular inoculum required to detect a steroidogenic response, increasing concentrations of granulosa cells (1, 2.5, and 5 x 105 viable cells/dish) were cultured for up to 72 h under serum-free conditions in the absence of treatment or in the presence of FSH (4 mIU/ml), with or without IGF-I (100 ng/ml). As shown (Fig. 6AGo), treatment with FSH produced significant increments in progesterone accumulation in a cell density-dependent fashion. More importantly, cotreatment with IGF-I resulted in the amplification of FSH hormonal action regardless of the cell density employed (4.6-, 2.6-, and 2.9-fold increments, respectively; P < 0.02). The IGF-I effect was time dependent (Fig. 6BGo), revealing 7.5-fold (P < 0.02), 3.6-fold (P < 0.02), and 3.6-fold (P < 0.06) increments at 24, 48, and 72 h, respectively. In contrast, the IGF-I effect proved independent of the FSH dose employed (Fig. 6CGo), revealing 4-, 4-, 4-, and 3.5-fold increments at 0, 0.4, 1.2, and 4.0 mIU/ml, respectively. Finally, 2 µg/ml doses of both IGFBP-2 and IGFBP-3 displayed potent antigonadotropic activity (43% and 47% inhibition, respectively), as assessed by FSH-supported accumulation of progesterone (Fig. 6DGo; P < 0.01 and 0.06, respectively).



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  		Figure 6. Steroidogenic characteristics of the mouse intraovarian IGF-I system: cultured granulosa cells. A, Granulosa cells (1, 2.5, and 5 x 105 viable cells/dish) were cultured for 72 h in the absence or presence of FSH (2 mIU/ml), with and without IGF-I (100 ng/ml). B, Granulosa cells (1 x 105 viable cells/dish) were cultured for the duration indicated in the absence or presence of FSH (2 mIU/ml) with and without IGF-I (100 ng/ml). C, Granulosa cells (1 x 105 viable cells/dish) were cultured for 72 h in the absence or presence of the indicated concentrations of FSH, with or without IGF-I (100 ng/ml). D, Granulosa cells (1 x 105 viable cells/dish) were cultured for 72 h in the absence of treatment or in the presence of FSH (2 mIU/ml) or FSH and IGFBP-2 (1 µg/ml). Thereafter, the medium content of progesterone was determined by RIA. Results represent the mean ± SE of three to five experiments.

 
Steroidogenic characteristics of the mouse intraovarian IGF-I system: cultured whole ovarian dispersates
To determine the degree to which the whole ovarian culture system could be miniaturized, increasing cellular densities of androstenedione-supplemented whole ovarian dispersates were cultured for 72 h in the absence or presence of FSH (4 mIU/ml) or FSH plus IGF-I (100 ng/ml). A stimulatory effect of FSH (5.9-fold increase) was apparent at a cellular density of 1000 viable cells/dish (Fig. 7AGo; not shown due to scale). The ability of IGF-I to amplify FSH hormonal action (2.7-fold increase; P < 0.13) was likewise apparent at a cellular density of 1000 viable cells/dish. Qualitatively comparable data were derived for the higher cell densities studied.



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  		Figure 7. Steroidogenic characteristics of the mouse intraovarian IGF-I system: cultured whole ovarian dispersates. A, Cells (0.1–10 x 104 viable cells/dish) were cultured for 72 h as described in Fig. 6AGo. B, Cells (1 x 105 viable cells/dish) were cultured for 72 h in the absence or presence of FSH (2 mIU/ml) or IGF-I (100 ng/ml), with or without androstenedione (2 x 10-7 M). C, Cells (1 x 104 viable cells/dish) were cultured for 72 h in the presence of increasing concentration of FSH (0–4 mIU/ml), with or without androstenedione (2 x 10-7 M). D, Androstenedione (2 x 10-7 M)-supplemented cells (1 x 105 viable cells/dish) were grown in the absence or presence of FSH (2 mIU/ml), FSH plus IGF-I (100 ng/ml), FSH plus IGFBP-3 (1 µg/ml), or FSH, IGF-I, plus IGFBP-3. Thereafter, the medium content of progesterone was determined by RIA. Results represent the mean ± SE of three experiments.

 
Using an optimal dose of FSH (4 mIU/ml), as defined in the previous experiment (Fig. 6CGo), whole ovarian dispersates (1 x 105 viable cells/dish) were cultured for 72 h in the absence of treatment or in the presence of FSH (4 mIU/ml) or FSH plus IGF-I (100 ng/ml), with or without androstenedione (2 x 10-7 M). The latter was chosen not only to serve as an aromatase substrate, but also for its ability to amplify FSH action (52). As shown (Fig. 7BGo), the provision of IGF-I substantially enhanced (2.5-fold increase) FSH hormonal action, an effect further augmented by the concurrent presence of androstenedione.

To assess the responsiveness of whole ovarian dispersates of mouse origin to FSH, whole ovarian dispersates (1 x 105 viable cells/dish) were cultured for 72 h under serum-free conditions in the presence of increasing concentrations of FSH (0–4 mIU/ml), with or without androstenedione (2 x 10-7 M). As shown (Fig. 7CGo), provision of androstenedione substantially augmented FSH action over a broad FSH dose range. Moreover, treatment of androstenedione-supplemented cells with increasing concentrations of FSH produced progressive increments in the accumulation of progesterone.

To examine the ability of IGFBP-3 to exert an antigonadotropic effect, androstenedione-supplemented whole ovarian dispersates were grown in the absence or presence of FSH (4 mIU/ml), FSH plus IGF-I (100 ng/ml), FSH plus IGFBP-3 (2 µg/ml), or FSH, IGF-I, and IGFBP-3. As shown (Fig. 7DGo), treatment with IGF-I substantially augmented FSH hormonal action, as assessed by the accumulation of progesterone. The IGF-I effect was partially blocked by the concurrent presence of IGFBP-3 acting, in all likelihood, to sequester some of the active ligand. However, IGFBP-3, applied by itself, displayed modest antigonadotropic activity (30% inhibition; P < 0.19) compatible with its ability to sequester endogenously generated IGF-I.

Cellular labeling studies: pregnenolone metabolism in mouse whole ovarian disperates
Delineation of the pathways of progestin metabolism in whole ovarian dispersates was accomplished by pulse labeling cells with [3H]pregnenolone after an initial culture period of 72 h in the absence or presence of FSH. Medium metabolites were extracted and analyzed by HPLC as described in Materials and Methods. As shown (Fig. 8Go), pregnenolone was metabolized to downstream products as a function of both time (left panel) and cell number (right panel). Using a small (3000 cells/dish) inoculum (Fig. 8Go, left panel), pregnenolone substrate disappeared as a function of time, coincident with the appearance of one major peak that coeluted with progesterone. However, 48 h were required to metabolize only 20% of the substrate. In contrast, all of the pregnenolone substrate disappeared by 48 h in cultures containing 1–3 x 105 cells/dish (Fig. 8Go, right panel). In these cultures, the major products coeluted with progesterone and 20{alpha}-dihydroprogesterone (Fig. 9Go), although other products were also apparent, as shown in Fig. 9Go. Importantly, however, no C19 or C18 steroids were detected. Under the conditions shown (Fig. 8Go), metabolism was somewhat elevated in FSH-treated cells. These experiments demonstrate that the mouse cultures contain functionally competent enzymes for the entire progestin cascade.



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  		Figure 8. Cellular labeling studies: characterization of pregnenolone metabolism in whole ovarian dispersates. Whole ovarian dispersates (0.03–3 x 105 viable cells/dish) were initially cultured for 72 h in the absence (broken lines) or presence of FSH (solid lines; 2 mIU/ml). Thereafter, media were removed, the cells were washed, and media containing [3H]pregnenolone substrate (plus FSH) were added for an additional period as indicated. Media were then collected, extracted, and HPLC fractionated as described in Materials and Methods. Each point is the mean ± SE of three to six values from three separate experiments. The left panel shows metabolism of substrate by 3000 cells/dish during 4, 24, or 48 h of incubation. The right panel shows metabolism of substrate after 48 h by 0.03–3 x 105 cells/dish.

 


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  		Figure 9. Cellular labeling studies: pattern of pregnenolone metabolism in whole ovarian dispersates. As described in Fig. 6Go, whole ovarian dispersates (1 x 105 viable cells/dish) were cultured for 72 h in the presence of FSH, after which [3H]pregnenolone was added for an additional 48 h. A representative HPLC chromatogram is shown. Elution times of standard steroids are indicated by arrows (Po, progesterone; Pn, 5{alpha}-pregnan-3{alpha}-o1,20-one; 20{alpha}, 20{alpha}-dihydroprogesterone; Pdiol, 5{alpha}-pregnan-3{alpha}a-o1,20-one; 20{alpha}a, 20{alpha}-dihydroprogesterone; Pdiol, 5{alpha}-pregnan-3{alpha},20{alpha}-diol).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The pattern of IGF-I and type I IGF receptor gene expression was identical in the rat (3, 4) and mouse (29). Similarly, neither species expressed IGFBP-1 at the level of the ovary (18, 53). However, a marked departure from the rat was noted when assessing the pattern of expression of IGFBP-2. In the rat ovary, IGFBP-2 mRNA was concentrated in the thecal-interstitial compartment of both the adult cycling (15) (Zhou, J., and C. A. Bondy, unpublished) and immature animal (17). The adult mouse ovary has been shown to be a site of IGFBP-2 gene expression (53). Our current studies of the immature mouse ovary localize IGFBP-2 mRNA primarily to the granulosa cell. Interestingly, the human ovary also expresses IGFBP-2 mRNA in both granulosa and thecal-interstitial cells (54), and the porcine ovary expresses IGFBP-2 primarily in granulosa cells, similar to the mouse (55). Only a modest IGFBP-3 mRNA signal could be detected in the immature mouse ovary (not shown), as shown previously in adult mice (53). Although the immature (noncycling) rat ovary may be a site of IGFBP-3 gene expression (22), it is the involuting corpus luteum of the adult cycling rat that is most prominent in this regard (56). This study did not address the possibility of IGFBP-3 mRNA expression in mouse corpora lutea. The ovarian expression of IGFBP-4 mRNA in the immature mouse ovary was minimal, in keeping with the reported failure to detect a signal corresponding for IGFBP-4 in whole ovarian material from adult mice (53). Findings in both immature mouse and rat suggest that IGFBP-4 mRNA is occasionally expressed by granulosa cells as well as by extrafollicular endothelial and fibroblastic cells (Zhou, J., and C. A. Bondy, unpublished observations). Although atretic follicles of adult cycling rats have been reported as a site of IGFBP-4 expression (18, 23, 57), no comparable data are available for the mouse model. IGFBP-5 mRNA, previously detected in the adult mouse ovary (53), was concentrated in granulosa cells of small follicles located in the cortical region of the mouse ovary. Yet another prominent site of expression was the germinal epithelium (Fig. 1Go). In this respect, no departure from the rat was noted, as IGFBP-5 mRNA has also been detected in the granulosa cells of a subset of follicles in the mature (24) and immature (Zhou, J., and C. A. Bondy, unpublished data) rat ovary. In summary, the cellular patterns of gene expression for components of the IGF-I system were similar in the immature rat and mouse ovary with respect to IGF-I and its receptor. However, significant differences were noted in the cellular pattern of IGFBP-2 gene expression, which in the mouse appears to occupy an intermediary position between those in the rat and human.

We also characterized the pattern of ovarian IGFBP expression in the mouse using cultured granulosa cells. As shown, untreated granulosa cells elaborated a relatively modest signal for a ligand-blottable 28- to 29-kDa doublet as well as a major single 24-kDa species. Size considerations, the immunoprecipitation data and the in situ localization data strongly suggest that the 28- to 29-kDa species is IGFBP-5. Size considerations and the immunoprecipitation data suggest that the 24-kDa IGFBP species is (presumptively nonglycosylated) IGFBP-4. The immunoprecipitation data also suggest that the 28-kDa IGFBP species is glycosylated IGFBP-4, as previously described for the rat granulosa cell (58). Finally, note was made of immunoprecipitatable IGFBP-2 (30-kDa). Consequently, the mouse and the rat ovarian paradigms differ in the types of IGFBPs elaborated by the granulosa cell. Although IGFBP-4 and IGFBP-5 appear to be shared by both species (58), the mouse granulosa cell appears to elaborate IGFBP-2 as well.

Previous studies have clearly established the ability of FSH to suppress the elaboration of rat granulosa cell-derived IGFBPs under both in vitro and in vivo circumstances (17, 21, 26). In part, this phenomenon appears to be transcriptional in nature (59). However, a posttranslational component entailing IGFBP-4- and IGFBP-5-directed proteases may also be involved (59, 60). Unexpectedly, treatment of cultured mouse granulosa cells with a maximal stimulatory dose of FSH had no effect on the relative accumulation of putative IGFBP-5 or IGFBP-4. In this respect, the mouse paradigm once again displays a marked departure from its rat counterpart. Provision of a maximal stimulatory dose of IGF-I proved up-regulatory for the accumulation of putative IGFBP-5, a phenomenon previously demonstrated for the rat (61). However, treatment with IGF-I led to inhibition of the accumulation of the 24-kDa IGFBP in the mouse; no comparable effect was established for the rat homolog (61). These findings suggest that both the pattern of expression and the pattern of regulation of IGFBPs differ markedly between mouse and rat granulosa cells.

In examining the operational characteristics of cultures of isolated mouse granulosa and whole ovarian dispersates, no major departures were noted compared with the corresponding paradigms in the rat. Specifically, both species are similar in their metabolism of radiolabeled pregnenolone (62), in their ability of IGF-I to amplify FSH action (63, 64, 65), and in their ability of representative IGFBPs to exert an antigonadotropic effect (19, 20, 22, 23, 56, 57, 66, 67). It would, therefore, appear that from an operational point of view, the mouse and rat granulosa cell systems are largely comparable. We have also demonstrated the feasibility of using small numbers of cultured mouse ovarian cells (e.g. an inoculum of 1000 viable cells/dish) without compromising the detection of alterations in the ambient concentrations of progesterone or radioactively labeled steroids.

The above notwithstanding, relatively minor differences were noted for the operational characteristics of mouse and rat ovarian cultures. First, whole ovarian dispersates (but not granulosa cells) of rat origin have previously been shown incapable of producing progesterone in response to treatment with FSH (68). Second, whole ovarian dispersates of mouse origin engaged in less robust steroidogenesis compared with rat counterparts (68, 69). Indeed, the overall progesterone output per cell is reduced for mouse compared with rat whole ovarian dispersates. Moreover, rat (unlike mouse) ovaries (1–3 x 105 cells/dish) are able to completely degrade progesterone within 2–4 h. Third, radiolabeling of mouse whole ovarian dispersates failed to yield [3H]androsterone by 48 h. This is not the case for rat whole ovarian dispersates, in which both androsterone and 5{alpha}-pregnanediol constitute dominant metabolites given a comparable period of culture (68, 69). Together, these findings suggest that the in vitro steroidogenic potential may be relatively attenuated in mouse compared with rat whole ovarian dispersates.

Taken together, our present findings indicate that the mouse intrafollicular IGF-I system differs from the rat paradigm in both the makeup and regulation of granulosa cell-derived IGFBPs as well as in the intensity and character of the steroidogenic process, thereby suggesting potential meaningful differences in ovarian physiology.


   Acknowledgments
 
The authors thank Ms. Cornelia T. Szmajda for her invaluable assistance with the preparation of this manuscript.


   Footnotes
 
1 This work was supported in part by NIH Research Grant HD-19998 (to E.Y.A.). Back

Received October 28, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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