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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 179-186
Copyright © 1998 by The Endocrine Society

Original Studies

Interferon-{gamma} and Activin A Promote Insulin-Like Growth Factor-Binding Protein-2 and -4 Accumulation by Human Luteinizing Granulosa Cells, and Interferon-{gamma} Promotes Their Apoptosis1

Nicholas A. Cataldo2, Victor Y. Fujimoto3 and Robert B. Jaffe

Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556

Address all correspondence and requests for reprints to: Dr. Robert B. Jaffe, Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California 94143-0556.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor (IGF)-binding proteins (IGFBPs) antagonize IGF and gonadotropin actions on granulosa cells. Human atretic follicles express IGFBP-2 in granulosa cells more strongly and contain higher levels of IGFBP-2 and IGFBP-4 than healthy follicles. We studied the effects of interferon-{gamma} (IFN{gamma}) and activin A, which decrease progesterone accumulation, on granulosa cell IGFBP production and apoptosis. Conditioned media from luteinizing granulosa cells cultured with IFN{gamma} or activin A and/or LH were subjected to ligand blotting; northern blots of total ribonucleic acid (RNA) from these cells were probed for IGFBP-2 and -4. Apoptosis was measured by in situ DNA end labeling. LH decreased medium IGFBP-2 to 21% of the control value. Although IFN{gamma} did not alter basal medium IGFBP-2, in the presence of LH it increased IGFBP-2 3.4-fold, with parallel changes in messenger RNA levels. Activin A also tended to increase medium IGFBP-2 in LH-treated cultures. In conditioned medium, IGFBP-4 was consistently decreased by LH, whereas both IFN{gamma} and activin A increased IGFBP-4 and decreased IGFBP-4 protease activity. Both LH and IFN{gamma} modestly stimulated IGFBP-4 messenger RNA levels. Follistatin antagonized the action of activin A, but not that of IFN{gamma}. IFN{gamma}, but not activin A, increased granulosa cell apoptosis. In conclusion, IFN{gamma} produced by activated lymphocytes may decrease endogenous IGF activity through stimulation of IGFBPs and may promote apoptosis of granulosa-lutein cells in vivo and, thus, luteal regression. Activin A similarly promotes IGFBP accumulation, but it does not promote apoptosis. (J Clin Endocrinol Metab 83: 179–186, 1998)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factors (IGF-I and IGF-II) promote ovarian follicular cell mitosis and differentiated function and can prevent granulosa cell (GC) apoptosis (1, 2). IGF action on the ovary can be antagonized by members of a family of binding proteins [IGF-binding proteins (IGFBPs)] (1). Of the first six IGFBPs identified by cloning (3), all except IGFBP-6 are expressed by human GC and are under differential regulation (1, 4, 5, 6, 7, 8). IGFBPs can decrease GC DNA synthesis and steroidogenesis in vitro, opposing the actions of IGFs and gonadotropins (9, 10, 11, 12). IGFBPs have also been implicated in follicular atresia and GC apoptosis, which occurs in atretic follicles, both in vivo and in vitro (2, 5, 13, 14, 15, 16). Follicular fluid levels of both IGFBP-2 and IGFBP-4 are higher in androgen-dominant, presumptively atretic, follicles than in estrogen-dominant, presumptively healthy ones (14, 15, 16).

Interferon-{gamma} (IFN{gamma}) is a cytokine produced by activated T lymphocytes and natural killer cells that has pleiotropic actions within the immune system (17). IFN{gamma} can also modulate the differentiated functions of epithelial cells. In cultured GC, IFN{gamma} inhibits the production of steroids and gonadotropin-dependent proteins, including LH receptor and inhibin (18, 19, 20), and induces Fas (CD95), a cell surface receptor that can transduce an apoptotic signal (21, 22, 23). In the human ovary, IFN{gamma} is present in follicular fluid at oocyte harvest for in vitro fertilization and is produced by mononuclear cells in preovulatory follicles and the corpus luteum (24, 25), but its biological role has not been established.

Activins are dimeric proteins produced by GC, which were first isolated as stimulators of pituitary FSH release (26). Activin A, a homodimer of ßA-subunits, can decrease steroidogenesis by luteinized granulosa or luteal cells in vitro (27, 28, 29). When administered systemically to rhesus monkeys, activin A disrupted folliculogenesis (30), and when injected under the ovarian bursa of gonadotropin-primed immature rats, it promoted follicular atresia (31).

Given the evidence that IFN{gamma} and activin A, like IGFBPs, inhibit GC steroidogenesis and may be implicated in GC apoptosis, we hypothesized that these two proteins might increase GC production of IGFBP-2 and IGFBP-4, the two IGFBPs that are increased in follicular fluid from atretic follicles. Upon finding that both IFN{gamma} and activin A promote accumulation by GC of these IGFBPs in vitro, we also examined their effects on apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Recombinant human (rh-) IFN{gamma} and rh-activin A were provided by the Research Reagents Program, Genentech (South San Francisco, CA). As IFN{gamma} is variably glycosylated, its concentration is stated in mass, rather than molar, units. Purified porcine follistatin was provided by Dr. Louis DePaolo, Whittier Institute (La Jolla, CA); its mol wt was taken as 35 kDa (26). Human LH was provided by the National Pituitary Agency. Complementary DNA (cDNA) probes for human IGFBP-2 and IGFBP-4 were provided by Dr. Shunichi Shimasaki, Whittier Institute, and the inserts were excised with appropriate restriction enzymes and gel purified before probe synthesis.

Cell culture

Granulosa cells were obtained at oocyte harvest from women undergoing in vitro fertilization after treatment with a GnRH agonist (leuprolide acetate or nafarelin acetate), human menopausal gonadotropins (Pergonal and/or Metrodin, Serono Laboratories, Randolph, MA), and hCG and purified as described previously (7, 29). This purification includes incubation with 0.1% hyaluronidase in a tissue culture flask for 30 min at 37 C, followed by centrifugation over 50% Percoll. Institutional review board exemption was obtained for the use of these otherwise discarded cells. For studies of IGFBP accumulation in conditioned medium, 2–5 x 105 viable granulosa cells, as determined by trypan blue exclusion, were seeded in 1.0 mL defined medium 7F on 24-well plates precoated with 1 µg/cm2 human plasma fibronectin (Boehringer Mannheim, Indianapolis, IN). Medium 7F consists of high glucose DMEM-Ham’s F-12 (1:1; H-21-F-12, Cell Culture Facility, University of California, San Francisco) supplemented, as previously described (7), with insulin (2 mg/L), transferrin, sodium selenite, aprotinin, L-glutamine, penicillin, streptomycin, and fungizone. Each experiment contained granulosa cells from one or two patients. The day after plating, media were replaced with 1.0 mL medium 6F (7F medium with insulin omitted) and test substances, including LH, IFN{gamma}, activin A, and follistatin. Conditioned media were harvested 3–5 days later, clarified by centrifugation, and stored at -70 C for subsequent ligand blot analysis. For ribonucleic acid (RNA) studies, 4–8 x 105 granulosa cells were plated on 6-cm plates in H-21-F-12 supplemented with 10% FCS, L-glutamine, penicillin, streptomycin, and fungizone. Established 4- to 5-day cultures were treated with test substances for 2 days before extraction of total cellular RNA.

Ligand blotting

Conditioned media were concentrated 10- to 20-fold by centrifugation through BSA-pretreated Centricon-10 microconcentrators (10 kDa exclusion; Amicon, Beverly, MA) and subjected to SDS-10% PAGE and ligand blotting as previously described (14, 32). Each lane on a gel contained the entire medium concentrate from one well of a cell culture plate. Media from parallel wells in an experiment were analyzed in parallel lanes on the same blot, along with prestained protein molecular mass markers (14–200 kDa; Life Technologies, Gaithersburg, MD), human seminal plasma as a reference for IGFBP-2 and IGFBP-4 (33), and human midtrimester amniotic fluid as a reference for IGFBP-1 (34). The gels were electroblotted to nitrocellulose filters, and the filters were blocked with 1% BSA and incubated overnight at 4 C with 1 µCi (37 kilobecquerels) [125I] 3-iodotyrosyl-IGF-I (Amersham, Arlington Heights, IL; 74 terabecquerels/mmol), then washed and exposed to autoradiography film.

Immunoprecipitation

IGFBPs were identified by immunoprecipitation and subsequent ligand blotting of conditioned media as previously described (14). Rabbit antisera (Upstate Biotechnology, Lake Placid, NY) to IGFBP-1 (<0.5% cross-reactivity with IGFBP-2, -3, -4, and -5), IGFBP-2 (<0.5% cross-reactivity with IGFBP-1, -3, -4, and -5), IGFBP-4 (50% cross-reactivity with IGFBP-2 but <1% cross reactivity with IGFBP-1, -3, and -5), and IGFBP-5 (<0.5% cross-reactivity with IGFBP-1 and -4; <0.1% cross-reactivity with IGFBP-2 and -3) were used at 1.5–4 µL/tube containing a 50-µL suspension of protein A-bearing fixed staphylococci (Pansorbin, Calbiochem, La Jolla, CA).

Immunoblotting

Western blots prepared as described above were incubated with 3% Nonidet P-40 in Tris-buffered 0.15 mol/L NaCl, pH 7.4 (TBS), blocked with 1% BSA in TBS, and then incubated with IGFBP antiserum at a 1:1000 dilution in TBS overnight at 22 C. The blots were washed for 5 min in 0.1% Tween-20 in TBS and incubated for 2 h at 22 C with a 1:1500 dilution of donkey anti-rabbit IgG Fab coupled to horseradish peroxidase. IGFBPs were detected with ECL chemiluminescence reagents (Amersham).

IGFBP-4 protease assay

Conditioned medium samples were incubated at 37 C for 24 h with rh-IGFBP-4 (Austral Biologicals, San Ramon, CA) covalently cross-linked to [125I]IGF-II. The reaction was stopped by adding SDS sample buffer, and the samples were subjected to 12% PAGE. The gel was dried and exposed to film, and levels of the 18-kDa proteolytic fragment were compared among medium samples from cells exposed to various treatments (35).

Northern analysis

Granulosa cell total RNA was extracted by the single step acid-guanidinium-phenol method (36) and quantified by absorbance at 260 nm (A260). Ratios of A260:A280 were greater than 1.7. Equal aliquots of total RNA from cultures derived from a single batch of granulosa cells exposed to different treatments in parallel were subjected to electrophoresis through 1.2% agarose gels containing 2 mol/L formaldehyde and capillary transferred to Nytran (Schleicher and Schuell, Keene, NH) in 0.3 mol/L sodium citrate-3 mol/L NaCl. UV cross-linked filters (Stratalinker, Stratagene, La Jolla, CA) were prehybridized at 68 C for 20 min in Quikhyb solution (Stratagene), hybridized for 60–90 min at 68 C with 2 x 107 cpm denatured IGFBP cDNA probe synthesized by random priming using [{alpha}-32P]deoxy-CTP (Amersham; 111 terabecquerels/mmol), and purified by ethanol precipitation. After hybridization, filters were washed and exposed to film. Blots were stripped and sequentially hybridized with labeled cDNA probes for each IGFBP and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; American Type Culture Collection, Rockville, MD), as a control for RNA loading.

Determination of granulosa cell apoptosis

Granulosa cells were cultured on multiwell glass microscope slides (LabTek, Nunc, Naperville, IL) in serum-supplemented medium and treated for 3–5 days with combinations of LH and IFN{gamma} or activin A. The cells were then fixed with phosphate-buffered 4% paraformaldehyde or Histochoice (Amresco, Solon, OH). DNA end labeling with digoxigenin-deoxy-UTP and terminal transferase followed by immunocytochemical staining with peroxidase-coupled antidigoxigenin antibody and diaminobenzidine were carried out with the reagents supplied in the Apoptag kit (Oncor, Gaithersburg, MD) according to the manufacturer’s instructions, except that Tris was substituted for phosphate in the wash buffer. After light counterstaining with hematoxylin, nuclei that stained brown were scored as positive for apoptosis, and those that stained blue were scored as negative. At least 300 cells/treatment condition in at least three x200 microscope fields were scored, and an apoptotic index was calculated as the percentage of cells in each treatment condition that were scored positive.

Progesterone accumulation

Granulosa cells were treated with IFN{gamma} and/or LH in 6F medium for 2 days, and medium progesterone was assayed with a RIA kit from Diagnostic Systems Laboratories (Webster, TX).

Quantitation of cellular DNA

Cultured cells were frozen at -20 C and later thawed for DNA quantitation with Hoechst 33258 (Hoefer Pharmacia Biotech, San Francisco, CA) (37). Cells were lysed in 1 mol/L NH4OH-0.2% Triton X-100 for 15 min at 37 C and neutralized with an equal volume of 1 mol/L KH2PO4, and the lysate was added to 2 mL 0.2 mol/L NaCl, 0.01 mol/L Tris-Cl, and 1 mmol/L sodium ethylenediamine tetraacetate, pH 7.4, containing 0.1 µg/mL Hoechst 33258. After excitation at 365 nm, fluorescence emission was measured at 460 nm with a DyNA Quant fluorometer (Hoefer Pharmacia) and compared with that of a calf thymus DNA standard. The DNA content of treated wells was compared to that in control untreated wells.

Data analysis

Autoradiograms were analyzed by integrated laser densitometry. For each autoradiogram, ratios of densitometric quantitation of IGFBP-2 results from treatment and control lanes derived from each cell culture experiment were expressed as a percentage, log transformed, and compared to the null hypothesis of no treatment effect by Student’s t test. As IGFBP-4 bands were frequently too faint for accurate quantitation by densitometry, their relative intensities on a ligand blot were qualitatively scored for each experiment, and the rank scores were compared with Friedman’s test and Student-Newman-Keuls post-hoc testing. Northern blots for each IGFBP messenger RNA (mRNA) were analyzed by densitometry; data derived from parallel lanes on a blot representing different treatment conditions were normalized to the signal intensity of GAPDH mRNA in the same lane and compared in the same fashion as the IGFBP-2 signal intensities on ligand blots. In studies of apoptosis induction, ratios of the apoptotic index in treated cells to that in control cells in each experiment were log transformed and compared to the null hypothesis of no treatment effect by t test. In studies of progesterone accumulation, the mean DNA-normalized progesterone level in each replicate culture well was compared among treatments by ANOVA and Fisher’s protected least significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of IGFBPs in granulosa cell-conditioned medium

Ligand blotting of concentrated conditioned medium from human luteinizing GC revealed a prominent doublet of bands at 31–33 kDa, as reported previously (7). Both bands were specifically immunoprecipitated with antiserum to IGFBP-2 (Fig. 1AGo). These bands, but no smaller ones, were also apparent on immunoblotting for IGFBP-2 (not shown). In most experiments, ligand blots of medium also showed a fainter 24-kDa IGFBP, identified by immunoprecipitation as IGFBP-4 (Fig. 1BGo). A 27-kDa IGFBP, detectable in a minority of experiments, was identified as IGFBP-1 (Fig. 1CGo, lanes s and t). A fourth, diffuse IGFBP band, previously identified as IGFBP-3 (7), was occasionally seen at 37–43 kDa (not shown). Despite the abundant expression of IGFBP-5 mRNA by these GC (8), three approaches failed to detect IGFBP-5 in GC-conditioned medium: no bands on ligand blots of conditioned medium comigrated with purified human IGFBP-5 at 29 kDa; no IGFBP-5 was detected by immunoprecipitation and ligand blotting of conditioned medium (Fig. 1CGo, lane u), and no IGFBP-5 was detected by immunoblotting (not shown). The IGFBP-5 antiserum did specifically immunoprecipitate purified human IGFBP-5, as detected by ligand blotting (Fig. 1CGo, lanes n and o).



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  		Figure 1. Identification of IGFBPs in granulosa cell-conditioned medium. Samples of medium were immunoprecipitated with antiserum to IGFBP-2 (A), IGFBP-4 (B), and IGFBP-1 and IGFBP-5 (C), and the precipitates were solubilized and analyzed by ligand blotting. Lanes a–c, g–i, and s–v show ligand blots of medium analyzed without immunoprecipitation (L) or after immunoprecipitation with specific IGFBP antiserum ({alpha}2, anti-IGFBP-2; {alpha}4, anti-IGFBP-4; {alpha}5, anti-IGFBP-5; {alpha}1, anti-IGFBP-1) or nonimmune rabbit serum (N). Lanes d–f and j–l show human seminal plasma as a positive control for IGFBP-2 and IGFBP-4 (33); lanes m–o show purified human IGFBP-5; lanes p–r show human midtrimester amniotic fluid as a positive control for IGFBP-1 (34).

 
Effects of LH, IFN{gamma}, and activin A on IGFBP-2 and IGFBP-4 production

LH. LH (1 U/mL) decreased medium IGFBP-2 levels to 21% of the control value (P < 0.0001; Figs. 2Go and 3AGo), as reported previously (7). Similarly, LH decreased GAPDH-normalized steady state IGFBP-2 mRNA expression (1.3 kilobases) to 40% of the control value (P < 0.01; Fig. 4Go).



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  		Figure 2. Effects of LH, IFN{gamma}, and activin A on granulosa cell medium IGFBP accumulation and IGFBP-4 protease activity. A, Shown is a representative autoradiogram of a ligand blot of concentrated medium conditioned by granulosa cells for 4 days with LH (1 U/mL), IFN{gamma} (50 ng/mL), and/or purified porcine follistatin (FS; 2.9 nmol/L), added as shown. Lane a shows human seminal plasma (SP); lane b, human midtrimester amniotic fluid (AF). The exposure time was 3 days. B, Shown is an autoradiogram of a ligand blot of medium conditioned by granulosa cells treated for 5 days with LH (1 U/mL) and/or activin A (A; 1.5 nmol/L), added as shown. SP and AF are shown as molecular size markers. The exposure time was 3 days. C, Shown is an autoradiogram of a ligand blot of medium conditioned by granulosa cells treated for 3 days with added LH (1 U/mL), activin A (A; 1.5 nmol/L), and/or follistatin (FS; 4.0 nmol/L). Lane j shows SP and AF together as size markers. Similar results were obtained in a second experiment. The exposure time was 5 days. D, Shown is an autoradiogram of an IGFBP-4 protease assay electrophoretic gel. The rh-IGFBP-4-[125I]IGF-II complex (1.5 x 104 cpm) was incubated for 24 h at 37 C with samples of medium from GC treated with LH (1 U/mL), activin A (A; 30 ng/mL), and/or IFN{gamma} (I; 40 ng/mL) or without additions (C) as shown. C0, Medium from the same experiment as that in the adjacent C lane, with incubation at 37 C omitted; T0 and T24, tracer complex incubated without medium for the indicated time (hours). Protease activity parallels the intensity of a radiolabeled 18-kDa fragment (35). Lanes a–e and h–l each contain medium samples from a distinct experiment. The exposure time was 7 days.

 


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  		Figure 3. Effects of LH, IFN{gamma}, and activin A on medium IGFBP accumulation: summary. A, Shown are the IGFBP-2 levels in granulosa cell-conditioned medium, determined by ligand blotting and densitometry, as a percentage of the level in medium from the control untreated wells of the same experiment analyzed on the same ligand blot. Each shows the results from one cell culture experiment. The horizontal bars indicate geometric mean IGFBP-2 levels. Statistical significance is indicated as follows: #, P < 0.01 vs. control; +, P < 0.01 vs. LH alone. B, Shown are qualitative IGFBP-4 levels in conditioned media with each treatment condition, as determined by examination of ligand blots, relative to levels in media from control basal or LH-stimulated (*) cultures as indicated. Each symbol shows the results from one cell culture experiment. For the nine experiments that included treatments with LH and activin A, both alone and in combination, a significant treatment effect was demonstrated for LH as well as for activin A in both basal and LH-stimulated cultures [overall P < 0.001, by Friedman’s test, with post-hoc P < 0.05 vs. control, by Student-Newman-Keuls test, as shown (#)].

 


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  		Figure 4. Effects of IFN{gamma} and LH on granulosa cell expression of IGFBP-2 and IGFBP-4 mRNA. Top, Shown is a representative Northern blot of total RNA (18 µg) from granulosa cell cultures treated for 2 days as indicated (C or CTRL, control) and sequentially hybridized with labeled cDNA probes for IGFBP-2, GAPDH, and IGFBP-4. Each probe hybridized to a single mRNA band of the size indicated, consistent with prior reports (4, 5). Exposure times were: IGFBP-2, 26 h; GAPDH, 5 h; and IGFBP-4, 19 h. The graph shows in arbitrary densitometric units the relative intensities of the mRNA signals on this blot for each of the three mRNA species. Bottom, Shown for each treatment are the relative IGFBP-2 (left) and IGFBP-4 (right) mRNA levels expressed by cultured granulosa cells, determined by densitometry of Northern blots, normalized to GAPDH expression in the same lane on the same blot, and calculated as a percentage of the corresponding value in control untreated cultures. Each symbol shows the results from one blot, containing RNA from a separate cell culture experiment. The horizontal bars indicate geometric means. Statistical significance is shown as follows: *, P < 0.01 vs. control; +, P < 0.01 vs. LH alone.

 
IGFBP-4 consistently gave a weaker signal on ligand blots than IGFBP-2, and in 5 of 16 experiments, no IGFBP-4 could be detected in medium from either control or LH-treated cultures. In all of the remaining 11 experiments, IGFBP-4 was detectable in control medium and was decreased by LH, in 9 of these to undetectable levels (Figs. 2BGo and 3BGo; P < 0.05). By contrast, in 2 experiments, LH tended to increase IGFBP-4 mRNA expression (2.0 kilobases; Fig. 4Go).

IFN{gamma}. In basal cultures, IFN{gamma} (50 ng/mL) did not significantly alter medium IGFBP-2 levels (130% of the control value; P > 0.05). Under LH-stimulated conditions, however, IFN{gamma} increased mean IGFBP-2 levels to 340% of those with LH alone (P < 0.01). With IFN{gamma} and LH, IGFBP-2 was 81% of that in basal control medium (P > 0.05). A representative ligand blot is shown in Fig. 2AGo, and data from all experiments are summarized in Fig. 3AGo. In basal cultures, IFN{gamma} (40–50 ng/mL) had no effect on IGFBP-2 mRNA levels, but in LH-stimulated cultures, it reversed the inhibition of IGFBP-2 expression by LH (IFN{gamma} plus LH, 80% of the control value; P < 0.01 vs. LH alone; Fig. 4Go).

IFN{gamma} increased medium IGFBP-4 under both basal and LH-stimulated conditions in four of five experiments; in one experiment, no IGFBP-4 was detected with or without IFN{gamma} (Figs. 2AGo and 3BGo). The IFN{gamma}-induced increase in medium IGFBP-4, estimated to be 5-fold, may be attributable to the lower IGFBP-4 protease activity found in IFN{gamma}-treated compared with control conditioned medium (Fig. 2DGo, compare lanes i and k), in view of the failure of IFN{gamma} to decrease steady state IGFBP-4 mRNA levels (Fig. 4Go).

Activin A. In basal cultures, activin A (1.5 nmol/L; 43 ng/mL) had no significant effect on medium IGFBP-2 levels (120% of control; P > 0.05). In LH-stimulated cultures, activin A tended to increase medium IGFBP-2 (63% increase over LH treatment alone; P = 0.06). Variable results were noted in 10 experiments and are summarized in Fig. 3AGo; two ligand blots are shown in Fig. 2Go, B and C. In one experiment, activin A reversed the inhibition of IGFBP-2 by LH in dose-dependent fashion, with an ED50 of 0.25 nmol/L (Fig. 5Go).



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  		Figure 5. Dose dependence of the stimulation of medium IGFBP-2 levels by activin A. Granulosa cells were incubated for 3 days with LH (1 U/mL) and activin A at the indicated concentrations. IGFBP-2 levels in conditioned medium, measured by ligand blotting, are shown in arbitrary densitometric units. The ED50 for activin A was 0.25 nmol/L.

 
Activin A increased both basal and LH-stimulated medium IGFBP-4 (P < 0.05). Under basal conditions, activin A increased medium IGFBP-4 in 7 of 13 experiments (e.g. Fig. 2BGo), whereas no IGFBP-4 was detected in either control or activin-treated media in 3 of 13 experiments (e.g. Fig. 2CGo). In LH-stimulated cultures, activin A increased medium IGFBP-4 in 6 of 9 experiments (e.g. Fig. 2BGo), whereas no IGFBP-4 was detected under either treatment condition in the other 3 (Figs. 2CGo and 3BGo). As with IFN{gamma}, these increases, consistently estimated to be at least 2-fold, may be attributable to the lower IGFBP-4 protease activity found in activin-treated compared with control conditioned medium (Fig. 2DGo, compare lanes b and d).

Modulation by follistatin of activin A, but not IFN{gamma} action

Follistatin is a high affinity, neutralizing activin-binding protein (29, 38). Purified porcine follistatin antagonized the stimulation of medium IGFBP-2 accumulation by activin A in LH-treated cultures (Fig. 2CGo, compare lanes g and h with c and d). Although the effects of IFN{gamma} on IGFBP-2 and IGFBP-4 accumulation are similar to those of activin A, neutralization of activin by follistatin did not block the effect of IFN{gamma} on medium levels of either IGFBP-2 or IGFBP-4 (Fig. 2AGo, compare lanes g and h with e and f).

IFN{gamma}, but not activin A, promotes granulosa cell apoptosis

On seven slides of cultured GC derived from five separate experiments, IFN{gamma} (50 ng/mL) treatment increased the apoptotic index regardless of concurrent LH stimulation (Fig. 6Go). In the absence of LH, IFN{gamma} increased the mean apoptotic index 3-fold, from 3.2% to 9.4% (P < 0.02), whereas in its presence, IFN{gamma} increased this index 2.8-fold, from 2.4% to 6.7% (P < 0.05). In three parallel experiments, activin A (1.5 nmol/L) failed to alter the apoptotic index of either unstimulated or LH-stimulated GC (data not shown).



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  		Figure 6. Effect of IFN{gamma} on granulosa cell apoptosis. Cultured granulosa cells treated without (left panel) or with (right panel) LH (1 U/mL) were incubated with IFN{gamma} for 3–5 days and then fixed, the DNA was end-labeled with digoxigenin and immunostained (Apoptag kit), and the percentage of apoptotic cells was counted. For each slide, the apoptotic index in the control well without IFN{gamma} (CTRL) and that in the IFN{gamma}-treated well are shown by paired connected symbols. The mean effect of IFN{gamma} to increase apoptosis was statistically significant (P < 0.05) both without and with LH.

 
Cell number in each culture well at the end of an experiment was also estimated from its DNA content. Treatment with LH, IFN{gamma}, activin A, or combinations of LH and either IFN{gamma} or activin A had no significant effect on the cellular DNA content of spent cultures (Table 1Go).


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  		Table 1. Effects of experimental treatments on DNA content of cultured cells

 
Effect of IFN{gamma} on medium progesterone accumulation (Fig. 7Go)

Treatment of GC with IFN{gamma} at 1 ng/mL or greater significantly decreased basal medium progesterone levels, whereas IFN{gamma} at 10 ng/mL or greater significantly decreased medium progesterone under LH-stimulated conditions.



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  		Figure 7. Effect of IFN{gamma} on progesterone accumulation in granulosa cell-conditioned medium. Shown are the mean ± sd medium progesterone levels, expressed for each well as nanograms per µg cellular DNA remaining at the end of the experiment and normalized to the level in control medium without LH or IFN{gamma}. Each of quadruplicate wells was seeded with 105 cells and incubated for 58 h with IFN{gamma} at the indicated concentrations (in nanograms per mL), without (left) or with (right) 1 U/mL LH. *, P < 0.05 vs. no IFN{gamma}. Consistent results were obtained in three additional experiments with 40 ng/mL IFN{gamma}.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IGF axis, consisting of peptide ligands, receptors, binding proteins (IGFBPs), and IGFBP proteases, is a well described regulatory system in the ovary (1, 39). In the human, IGF-II is produced by granulosa cells, and IGF-I is produced by thecal cells; the production of both peptides is stimulated by gonadotropins, and both are present in follicular fluid (see Ref. 1 for review). GC also produce IGFBPs (1, 5, 6, 40, 41). Human GC express IGFBP-1, -2, -3, -4, and -5 mRNA and produce all of these proteins except IGFBP-5 (1, 5, 6, 7, 8, 15). By immunohistochemistry and in situ hybridization, IGFBP-2 has been selectively localized to GC of small antral follicles (5). Levels of IGFBP-2 in follicular fluid are consistently higher in androgen-dominant, presumptively atretic, follicles, than in estrogen-dominant, presumptively healthy, ones (14, 15, 16); in vitro, GC IGFBP-2 synthesis is decreased by hCG (7). Like IGFBP-2, IGFBP-4 levels in follicular fluid are higher in atretic than in healthy follicles (14, 15, 16); an IGFBP-4 protease found in estrogenic, but not androgenic, follicular fluid (35) may account for the lower IGFBP-4 levels in the former. These findings point to a possible association between IGFBP-2 and IGFBP-4 and GC atresia/apoptosis. The absence of IGFBP-5 in human GC-conditioned medium despite abundant expression of its mRNA (8) may be due to the action of an IGFBP-5 protease similar to that reported in the rat (41).

In the present studies, LH decreased IGFBP-2 protein accumulation and steady state mRNA expression to a similar degree. These findings suggest that LH regulates IGFBP-2 gene transcription. LH consistently decreased medium IGFBP-4, but it increased its steady state mRNA levels by 50%. The latter finding parallels the slightly greater expression of IGFBP-4 noted by in situ hybridization in dominant compared to small antral follicles (5). Taken together, these findings suggest that gonadotropins stimulate GC production of an IGFBP-4 protease, as may occur in healthy antral follicles (35).

As IFN{gamma} can decrease progesterone production by GC and can promote their expression of the apoptosis-signaling protein Fas (18, 19, 20, 21, 22, 23), we hypothesized that IFN{gamma} produced by lymphocytes, which invade the corpus luteum as the follicular basement membrane disintegrates at ovulation (42), may stimulate luteal production of IGFBP-2 and IGFBP-4 and may promote luteal regression by apoptosis. In a cell culture model, we found that IFN{gamma} indeed significantly increased medium IGFBP-2 accumulation in LH-stimulated, but not basal, cultures, completely reversing the inhibition of IGFBP-2 by LH. Similar to its effects at the protein level, IFN{gamma} increased steady state IGFBP-2 mRNA expression in LH-stimulated GC, suggesting that IFN{gamma} can act on IGFBP-2 gene transcription. IFN{gamma} stimulated medium IGFBP-4 levels in both basal and LH-stimulated cultures, but much weaker effects were seen on IGFBP-4 mRNA expression and only in basal cultures. Taken together, these findings suggest that the observed decrease in protease activity, rather than transcriptional control of gene expression, is responsible for the increase in medium IGFBP-4 after IFN{gamma} treatment.

As activin A can decrease both progesterone production and aromatase activity in luteinizing GC and may accelerate atresia of antral follicles (27, 28, 29, 30, 31), we hypothesized that activin produced by GC may promote accumulation of IGFBPs by these cells and may play a role in promoting their apoptosis. We found that like IFN{gamma}, activin A had no effect on IGFBP-2 levels in conditioned media in the absence of added LH, but tended to increase medium IGFBP-2 levels in LH-stimulated cultures. The failure of the latter effect to reach statistical significance and the heterogeneity of the observed response to activin A may reflect the inherent heterogeneity of the granulosa cells used in these studies, which derive from follicles of varying size and maturity from women of varying age and infertility diagnosis. These GC have been reported to show variable rates of apoptosis at the time of harvest (43). In the experiments in which activin A increased IGFBP-2 accumulation by LH-stimulated GC, such as that illustrated in Fig. 2CGo, its action was antagonized by follistatin and was dose dependent, with an ED50 similar to its affinity constant for type II activin receptors and its ED50 for inhibition of progesterone accumulation (29, 44). Unlike IFN{gamma}, activin A did not reverse the effects of LH on IGFBP-2 accumulation. As LH can stimulate follistatin production by GC (45), the action of activin on LH-stimulated GC may be muted by LH-stimulated follistatin production.

Activin A increased medium IGFBP-4 and decreased IGFBP-4 protease activity under both basal and LH-stimulated conditions. In a previous study of these GC, activin A did not significantly alter steady state IGFBP-4 mRNA levels (8). Taken together, these findings support the concept that activin A, like IFN{gamma}, increases medium IGFBP-4 by decreasing its proteolysis, rather than by transcriptional regulation.

Although the effects of IFN{gamma} on IGFBP-2 and -4 accumulation and steroidogenesis were similar to those of activin A, IFN{gamma} does not appear to act by stimulating GC activin production, as its effects on medium IGFBP and progesterone (data not shown) levels were not blocked by follistatin.

The finding that IFN{gamma} increased GC apoptosis in culture, even under LH-stimulated conditions, suggests a role for this cytokine in human granulosa-luteal cell apoptosis. IFN{gamma} may be an effector of the increase in apoptosis observed in the midluteal phase human corpus luteum, a time of ongoing LH stimulation (46). The involvement of a cytokine in both regulation of the intraovarian IGF system and apoptosis induction suggests a significant physiological role for lymphocytes and macrophages in the cyclic processes of follicular atresia and luteal regression, both of which involve apoptosis of ovarian cells (13, 46). Our finding that activin A does not promote apoptosis of human GC, at least in monolayer culture, suggests that the antisteroidogenic action of activin A on luteal cells does not result from apoptosis induction and argues against a role of activin in human luteal regression despite its ability to promote GC apoptosis in antral follicles in a rodent model (31).

Our studies do not allow a conclusion to be drawn as to whether IGFBPs mediate the effects of either IFN{gamma} or activin A on GC steroidogenesis or apoptosis. Although added IGF-I (neutralization of IGFBP) failed to reverse the inhibition of progesterone production by IFN{gamma} in a single experiment and inconsistently reversed the effects of activin A on progesterone (data not shown), only a study of the effects of direct addition of pure IGFBPs to cultured GC can answer this question. Although induction of Fas antigen by IFN{gamma} and its activation (21, 22, 23) may account for the promotion of apoptosis by this cytokine, this mechanism is unlikely to account for its inhibition of steroidogenesis, as Fas activation alone fails to lower basal medium progesterone (22) (our unpublished observations).


   Acknowledgments
 
Granulosa cells were generously furnished by the staff of the University of California, San Francisco In Vitro Fertilization Program and by Dr. Frank Polansky and Ms. Eve Shen, Nova In Vitro Fertilization (Palo Alto, CA). Purified human IGFBP-5 was generously provided by Dr. Sharron Gargosky, Department of Pediatrics, Oregon Health Sciences University (Portland, OR). [125I]IGF-II-rh-IGFBP-4 protease assay substrate was kindly made available by Drs. Yasmin Chandrasekher and Linda Giudice, Stanford University (Stanford, CA). The technical assistance of Ms. Carmelita Aguirre with the progesterone assays and that of Ms. Phuong Hoang with preparation of the cDNA inserts are greatly appreciated.


   Footnotes
 
1 This work was supported in part by an American Fertility Society-Serono Laboratories Research Grant in Reproductive Medicine and NIH Grant K08-HD-01141 (to N.A.C.). Presented in part at the 41st Annual Meeting of the Society for Gynecologic Investigation, Chicago, IL, March 1994, and at the symposium, The Life Cycle of the Ovarian Follicle, sponsored by Serono Symposia USA, Fort Lauderdale, FL, November 1995. Back

2 Present address: Department of Gynecology and Obstetrics, Stanford University Medical Center, 300 Pasteur Drive, Stanford, California 94305-5317. Back

3 Present address: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Washington, 4225 Roosevelt Way NE, Suite 305, Seattle, Washington 98105. Back

Received February 13, 1997.

Revised September 4, 1997.

Accepted September 16, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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