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Endocrinology Vol. 143, No. 6 2259-2267
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Follicle-Stimulating Hormone Amplifies Insulin-Like Growth Factor I-Mediated Activation of AKT/Protein Kinase B Signaling in Immature Rat Sertoli Cells

Shafiq A. Khan, Lilianne Ndjountche, Lauren Pratchard, L. J. Spicer and John S. Davis

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center (S.A.K., L.P.), Lubbock, Texas 79430; Veterans Affairs Medical Center, Women’s Research Institute, University of Kansas Medical Center (L.N., J.S.D.), Wichita, Kansas 67214; Department of Animal Science, Oklahoma State University (L.J.S.), Stillwater, Oklahoma 74078; and Olson Center for Women’s Health, Department of Obstetrics and Gynecology, University of Nebraska Medical Center (J.S.D.), Omaha, Nebraska 68198

Address all correspondence and requests for reprints to: Shafiq A. Khan, Ph.D., Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, Texas 79430. E-mail: . shafiq.khan{at}ttmc.ttuhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 µM), (Bu)2cAMP (40 µM), or forskolin (10 µM). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10–20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1–4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 µM), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SERTOLI CELL has a complete autocrine IGF-I system (1, 2, 3, 4, 5, 6). Rat and porcine Sertoli cells have been shown to secrete IGF-I in vitro (7, 8, 9). Localization of IGF-I by immunohistochemistry and in situ hybridization in rodent and porcine testes during postnatal development indicated that the levels of IGF-I are highest in Sertoli cells in newborn animals and gradually decrease and are very low in Sertoli cells in adult animals (10, 11, 12, 13). IGF-I receptors have also been shown to be present in rat and porcine Sertoli cells (3, 4, 5, 6). In addition, Sertoli cells secrete IGF-binding proteins (IGF-BPs), predominantly IGF-BP3, which inhibits the effects of exogenous IGF-I on Sertoli cells in vitro (14, 15, 16). Thus, the Sertoli cell contains the necessary components for the synthesis and reception of IGF-I and the modulation of IGF-I action. Furthermore, the Sertoli cell IGF-I system may be regulated by FSH. Both in vivo and in vitro studies demonstrate that FSH stimulates IGF-I production in Sertoli cells while it inhibits secretion of IGF-BP3 by immature Sertoli cells in vitro (15, 16, 17). In addition, other hormones, such as GH and thyroid hormone, and locally produced cytokines may be involved in regulation of the IGF-I system in Sertoli cells (1, 3, 18, 19).

FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. IGF-I has been shown to exert multiple effects on cultured immature Sertoli cells, such as stimulation of proliferation, lactate production, glucose transport, transferrin production, and secretion of plasminogen activator (4, 20, 21, 22, 23, 24, 25). On the other hand, IGF-I inhibits FSH-dependent aromatase activity in immature rat Sertoli cells (26). Homozygous IGF-I-null mutant male mice are infertile with reduced serum T levels, reduced testicular size, and spermatogenesis only at 18% the normal level (12). These studies have suggested that IGF-I plays an important role in the in vivo proliferation and differentiation of Sertoli cells (20, 27, 28, 29).

The actions of IGF-I on target cells are mediated primarily by the type I receptor, which is coupled to multiple intracellular signaling cascades (30). Activation of the IGF-I receptor causes activation of PI3K, which leads to activation of serine-threonine kinase, AKT kinase, or protein kinase B (PKB) (31). The AKT/PKB pathway has been shown to be involved in the effects of IGF-I on cell survival and proliferation (32). Furthermore, activation of the IGF-I receptor also leads to activation of the p21ras pathway, resulting in activation of the ERK members of the MAPK pathway (33) that have been implicated in the effects of growth factors on cell survival and proliferation (34).

Recent studies in granulosa cells have indicated a role for IGF-I-stimulated PI3K-AKT kinase signaling pathway in cell survival (35, 36). Furthermore, FSH has been shown to activate AKT kinase in rat granulosa cells (37). Despite an established role for the IGF-I system in the development and function of immature Sertoli cells, the identity of the signaling cascades involved in a multitude of IGF-I effects on Sertoli cells and the effects of FSH, if any, on IGF-I signaling in these cells, remain unknown. The present study was carried out to investigate the effects of IGF-I on PI3K/AKT kinase signaling in immature rat Sertoli cells and to determine whether FSH influences the effects of IGF-I on this signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and animals
Recombinant human FSH was obtained from NIDDK (Bethesda, MD). Human recombinant IGF-I and human recombinant IGF-BP3 were purchased from R&D Systems (Minneapolis, MN) and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively. (Bu)2cAMP, 8-bromo-cAMP, PI3K inhibitor (LY 294002), PKA inhibitor (H89), collagenase (type 1), and deoxyribonuclease were purchased from Sigma (St. Louis, MO). Anti-phospho-AKT (no. 9271L), anti-AKT (no. 9272), anti-CREB (no. 9192), and anti-phospho CREB (no. 9191L) antibodies were purchased from Cell Signaling Technology (Beverly, MA).

All incubations and cell cultures of immature Sertoli cells were performed in Eagle’s MEM with Earle’s salts and 0.1 mM of the following amino acid supplements: L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and L-glycine. The medium also contained 4 mM L-glutamine, 2.5 g/liter NaHCO3, 1.5 mM HEPES, 50 U/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml gentamicin (Life Technologies, Inc., Grand Island, NY).

Immature (10-d-old) male Wistar Crl:(W1)BR rats were obtained from Charles River Laboratories, Inc. (Indianapolis, IN), and maintained with the mothers under standard conditions. All experimental protocols were approved by the institutional animal care and use committee of the Texas Tech University Health Sciences Center.

Isolation and culture of Sertoli cells
Immature Sertoli cells were isolated from 10-d-old rats using a modification of the method of Dorrington and Armstrong (38) as described previously (39). Briefly, the decapsulated testes were incubated for 15 min in culture medium containing 0.25 mg collagenase/ml. The seminiferous tubules were allowed to settle at unit gravity for 5 min, and the supernatant containing the suspension of interstitial cells was removed. The seminiferous tubules were then minced with scissors, and the tubular pieces were incubated with collagenase (1 mg/ml) and deoxyribonuclease (100 µg/ml) for 1 h at 37 C, followed by mild agitation by using a Pasteur pipette. The tubular pieces were washed extensively to remove peritubular cells, and the Sertoli cell aggregates were placed in 50-ml tubes and dispersed by gentle homogenization using a Kontes pellet pestle (Fisher Scientific, St. Louis, MO), followed by filtration through B-D Falcon cell strainers (nylon mesh size, 70 µm). The cells were plated in six-well culture plates (2 x 106 cells/well) or in 24-well plates (3 x 105 cells/well) and cultured at 37 C in a humidified atmosphere of 5% CO2 and 95% air in serum-free medium. In the experiments to study the effects of different treatments on the morphology and survival of immature Sertoli cells, the cells (3 x 105 cells) were cultured on sterile glass coverslips placed in six-well plates. After 72 h, the cells were extensively washed to remove unattached germ cells. The cells were cultured for additional 4 d in the serum-free medium, with medium changed after every 48 h. The cells were then treated with different concentrations of FSH and IGF-I for specific time periods to determine their effects on DNA synthesis, estrogen production and morphology, and signal transduction mechanisms.

DNA synthesis
Sertoli cells cultured in 24-well plates were treated in triplicate with FSH (100 mU/ml) in the presence or absence of IGF-I (50 ng/ml) for 18 h. The cells were then washed and incubated for an additional period of 4 h in the presence of [3H]thymidine (1 µCi/ml). The cells were lysed, and the incorporation of [3H]thymidine into DNA was determined as described previously (40).

Aromatase activity
After a 7-d culture in serum-free medium in 24-well plates, the Sertoli cells were treated in triplicate with FSH (100 mU/ml) in the presence or absence of IGF-I (50 ng/ml) for 24 h in the presence of 19-hydroxyandrostenedione (2.5 nM) as described previously (39). The conditioned media were collected, and the E2 levels were determined using an RIA kit (Diagnostic Products, Los Angeles, CA).

Morphology and cell survival
The cells cultured on coverslips were treated with control medium, the PI3K inhibitor LY 294002 (10 µM), or IGF-BP3 (5 µg/ml) for 24 h. The cells were fixed in freshly prepared 4% paraformaldehyde in PBS (pH 7.2). The cells were then incubated with 4',6-diamidino-2 phenylindole (DAPI; 0.5 ng/ml; Sigma) for 30 min, followed by washing in PBS (containing 0.1% Tween 20) for 8 min and in distilled water for 2 min. Fluorescence was observed with a Carl Zeiss Axiovert microscope, using an LD Achroplan x40 objective and standard UV filters to detect DAPI staining (Chroma Technology Corp., Brattleboro, VT). To monitor the changes in the morphology of Sertoli cells after various treatments, the cells were observed by phase contrast microscopy. The images were recorded electronically using Image Pro Plus software. The presence of pyconotic nuclei, nuclear fragmentation, or blebbing was used to identify apoptotic Sertoli cells after various treatments.

In a separate set of experiments the cells were cultured in triplicate in six-well plates in the presence of above inhibitors for 24 h. The cells were then washed, and total protein and DNA were extracted using TRIzol reagent (Life Technologies, Inc.) according to the instructions of the manufacturers. Total DNA content was determined spectrophotometrically, and the total proteins were determined using Bradford reagents (Bio-Rad Laboratories, Inc., Hercules, CA). For evaluation of oligonucleosome formation, genomic DNA from Sertoli cells treated with LY 294002 (10 µM) or IGF-BP3 (5 µg/ml) was prepared as described by Tilly et al. (41). Genomic DNA (10 µg) and a DNA ladder (1 µg; 0.5–10 kb; New England Biolabs, Inc., Beverly, MA) were electrophoresed in 2% agarose gels and detected by ethidium bromide staining.

Western blotting
To determine the effects of FSH and IGF-I on immunoreactive levels of total or phosphorylated forms of AKT (Ser473) or CREB (Ser133) proteins, immature Sertoli cells were cultured in six-well plates (2 x 106 cells/well) as described above. The cells were then washed and treated with various concentrations of FSH, IGF-I, or a combination of both for different time periods. For some experiments, the cells were preincubated in the presence of PI3K inhibitor (LY 294002; 10 µM), a PKA inhibitor (H89; 10 µM), or IGF-BP3 (5 µg/ml) for 30 min before the addition of FSH or IGF-I. At the end of the treatment period, the cells were washed with ice-cold PBS and lysed with ice-cold cell lysis buffer [20 mM Tris-HCl (pH 7.4), containing 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin; Cell Signaling Technology, Beverly, MA]. The lysis buffer was supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany) that inhibits a broad spectrum of proteases. The lysed cells were scraped from the plates, collected in Eppendorf tubes, and stored frozen at -80 C. Protein concentrations in cell lysates were determined using a DC protein assay kit (Bio-Rad Laboratories, Inc.). The cell lysates (15 µg protein) were subjected to SDS-PAGE in 10% gels and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The membranes were blocked overnight at 4 C in 50 mM Tris (pH 7.5), containing 0.15 M NaCl, 1 mM EDTA containing 0.05% Nonidet P-40, and 1% BSA. The blots were then incubated with specific antibodies that cross-react with total proteins or phosphorylated forms of AKT (Ser473, p-AKT; 1:1,500) and CREB (Ser133, p-CREB; 1:5,000) proteins for 2 h at room temperature. After washing, the blots were incubated with appropriate antirabbit IgG coupled to HRP (dilution 1:10,000) for 1 h. The blots were then developed in ECL Plus mixture (Amersham Pharmacia Biotech, Piscataway, NJ) for 1–10 min, exposed to x-ray film (Kodak Biomax Film, Eastman Kodak Co., Rochester, NY), and visualized by autoradiography. The relative intensities of specific protein bands were determined by densitometric scanning of images using a BioImage Visage 2000 (BioImage, Ann Arbor, MI) computer-assisted image analysis system. The ratios of intensities of phosphorylated proteins and total proteins were calculated to determine the extent of activation of specific kinases.

Determination of IGF-I and IGF-BPs in Sertoli cell-conditioned medium
Medium samples collected for IGF-I and IGF-BP assessment were ultrafiltrated using Centricon concentrators with a molecular weight limit of 3000 (Amicon, Inc., Beverly, MA) as previously described (42) with the following modifications. Briefly, 4 µl 0.25 g/ml BSA were added to 400 µl of the spent medium (to maximize recovery of IGF-BPs) and placed inside the sample reservoir of the concentrator and centrifuged at 5322 x g for approximately 80 min. The spent media were concentrated 7- to 13-fold. Concentrated medium samples were assayed for IGF-I after acid-ethanol extraction (16 h at 4 C) by RIA as previously described (43, 44). Increasing volumes of concentrated-extracted media gave displacements in binding parallel to the standard curve. The intraassay coefficient of variation for the IGF-I RIA was 9.7%. The sensitivity of the IGF-I RIA, defined as 95% of total binding, averaged 5.0 pg/tube.

The concentrated medium samples were assessed for IGF-BP activity based on molecular weight using one-dimensional, nonreducing, SDS-PAGE as described previously (42). Briefly, 12.5 µl concentrated culture medium were mixed with 12.5 µl Laemmli sample buffer (Bio-Rad Laboratories, Inc.). After heat treatment (3 min at 100 C) to denature the proteins, samples were centrifuged at 4700 x g for 3 min and loaded into wells of 12% polyacrylamide gels (15 lanes/gel). All medium samples from a single experiment were run on a single gel, and four gels were run in a single electrophoresis chamber. After electrophoresis, the proteins were electrophoretically transferred to nitrocellulose paper (Midwest Scientific, St. Louis, MO) for 2.5–3.0 h. Each nitrocellulose paper was then labeled with a mixture (1:1) of [125I]IGF-I and [125I]IGF-II (~15,000 cpm/0.1 ml; total volume, 6 ml) and placed on a rocking platform at 4 C overnight. The next day, the nitrocellulose blots were washed, dried, and exposed to x-ray film for 10 d at -80 C. At the end of the 10-d period, x-ray films were developed, and individual bands were densitometrically analyzed using a Molecular Analyst (Bio-Rad Laboratories, Inc.). IGF-BP activity was expressed as arbitrary densitometric units per 400 µl culture medium. IGF-BP-3, -4, and -5 bands were scanned, and the resultant arbitrary densitometric units for each IGF-BP were used to calculate the intergel coefficient of variation, which averaged 14 ± 5%.

Statistical procedures
All experiments were performed at least twice using a different cell preparations with identical results. The data are presented for representative experiments. The differences in the incorporation of labeled thymidine and estrogen production after various treatments (Fig. 1Go) were analyzed by t test.



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  		Figure 1. Effects of FSH and IGF-I on DNA synthesis (A) and E2 production (B) in immature rat Sertoli cells. The cells were treated with FSH (100 mIU/ml), IGF-I (50 ng/ml), or a combination of both (F+I) for 18 h (A) or 24 h (B). Incorporation of [3H]thymidine (A) was determined in the subsequent 4-h period, and E2 content in the incubation media (B) was determined by RIA.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I has previously been shown to stimulate DNA synthesis and to inhibit FSH-dependent aromatase activity in immature rat Sertoli cells (4, 26). To ascertain the biological relevance of the current studies, we first investigated the effects of IGF-I on DNA synthesis and aromatase activity in Sertoli cells. Sertoli cells were cultured for 7 d in serum-free medium, washed, and treated with FSH (100 mU/ml), IGF-I (50 ng/ml), or a combination of both for 18 h. The amount of [3H]thymidine incorporation into DNA was then determined during the subsequent 4-h period. In parallel experiments, the cells were treated with FSH and IGF-I for 24 h in the presence of androgen substrate, 19-hydroxyandrostenedione. The conditioned media were collected and analyzed by RIA for E2. As shown in Fig. 1AGo, both FSH and IGF-I caused a significant stimulation (1.5- and 6.2-fold, respectively) of DNA synthesis (P < 0.05; n = 3) in immature Sertoli cells, and the effects of the two agonists were additive. FSH caused a significant (~20 fold; P < 0.05; n = 3) stimulation of E2 production from androgen substrate in immature Sertoli cells (Fig. 1BGo). Although IGF-I alone had no effect on E2 production, it significantly inhibited (~40%; P < 0.05; n = 3) FSH-stimulated E2 production. These data demonstrate that the cultures of rat Sertoli cells used in the current study provide a relevant basis for the examination of cellular signal transduction mechanisms in response to IGF-I and FSH, alone or in combination.

Next, we investigated the effects of IGF-I and FSH on AKT phosphorylation in Sertoli cells. The cells were treated with various concentrations of IGF-I or FSH for 10 min, the cells were lysed, and total cellular proteins were analyzed for p-AKT and total AKT proteins by Western blotting. As shown in Fig. 2Go (top panel), IGF-I caused a significant concentration-dependent accumulation of p-AKT in rat Sertoli cells. FSH, on the other hand, had no effect on p-AKT during the 10-min treatment period (Fig. 2Go, middle panel). To investigate whether FSH influences the effects of IGF-I on p-AKT, the cells were incubated with increasing concentrations of FSH for 10 min, followed by incubation with IGF-I (100 ng/ml) for 10 min. As shown in Fig. 2Go (lower panel), FSH caused a concentration-dependent increase in the levels of p-AKT in IGF-I-stimulated Sertoli cells. On the average, IGF-I caused a 3.80 ± 0.48-fold (mean ± SEM; n = 10) increase (P < 0.05) in the phosphorylation of AKT after 10 min of treatment. Pretreatment with FSH for 10 min caused a further 1.87 ± 0.19-fold (mean ± SEM; n = 9) increase (P < 0.05) in AKT phosphorylation in response to IGF-I.



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  		Figure 2. Effects of IGF-I and FSH on phosphorylation of AKT in immature rat Sertoli cells. The cells were treated with different concentrations of IGF-I (top panel) and FSH (middle panel) for 10 min. In addition, the cells were incubated with different concentrations of FSH for 10 min, followed by incubation with IGF-I (100 ng/ml) for 10 min (lower panel). Cell lysates (15 µg total protein) were analyzed by Western blotting using specific antibodies against p-AKT or total AKT (AKT) protein. The numbers beneath each panel show the relative differences in the ratios of p-AKT and AKT proteins after different treatments, as determined from densitometric analyses of individual bands.

 
To determine whether the synergistic effects of FSH on IGF-I-stimulated phosphorylation of AKT (Fig. 2Go) were mediated by cAMP-dependent signaling mechanisms, Sertoli cells were incubated with FSH, forskolin, 8-bromo-cAMP, or (Bu)2cAMP for 10 min, followed by incubation with IGF-I for 10 min. The cells were then lysed and analyzed for levels of p-AKT. As shown in Fig. 3AGo, FSH, forskolin, or cAMP analogs, when added alone, had no effect on p-AKT; however, each of these treatments synergized with IGF-I to stimulate AKT phosphorylation. Under the same experimental conditions, FSH and forskolin caused a significant increase in the levels of p-CREB, whereas IGF-I had no effect on phosphorylation of CREB (Fig. 3BGo). To determine whether the synergistic effects of FSH on IGF-I-mediated activation of AKT were mediated by PKA, we studied the changes in the levels of p-AKT in Sertoli cells in response to FSH and cAMP in the presence of H89, a specific PKA inhibitor. As shown in Fig. 3CGo, H89 (10 µM) failed to block the synergistic effects of FSH on IGF-I-stimulated phosphorylation of AKT.



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  		Figure 3. Effects of forskolin and cAMP analogs on phosphorylation of AKT (A) and CREB (B) proteins in immature rat Sertoli cells. A, The cells were incubated in the presence of forskolin (10 µM), (Bu)2cAMP (db-cA; 40 µM), 8-bromo-cAMP (8b-cA; 40 µM), and FSH (0.9 IU/ml) for 10 min, followed by treatment with IGF-I (100 ng/ml) for 10 min. The cell lysates were then analyzed for p-AKT and AKT proteins by Western blotting. B, The levels of phosphorylated CREB (p-CREB) and total CREB (CREB) were analyzed in total cell lysates from Sertoli cells treated with IGF-I (I), FSH (F), forskolin (Fo), or a combination of FSH plus IGF-I (F+I) or forskolin plus IGF-I (Fo+I). C, Sertoli cells were preincubated with PKA inhibitor (H89; 10 mM) for 30 min, followed by treatment with IGF-I (I), FSH (F), or FSH and IGF-I (F+I) as described above, and the levels of p-AKT and total AKT were determined by Western blotting. Further details are given in Fig. 2Go.

 
To investigate whether extended incubation of Sertoli cells with FSH would lead to activation of AKT, Sertoli cells were treated with FSH (0.9 U/ml) for 10, 30, 60, and 240 min. Total cellular proteins were then analyzed by Western blotting as described above. As shown in Fig. 4AGo, FSH caused a significant accumulation of p-AKT in Sertoli cells after 30 min of incubation, which reached a maximum at 60 min and remained elevated in cells treated with FSH for 4 h. Additional experiments indicated that maximum stimulation of AKT phosphorylation was achieved 2 h after treatment with FSH (data not shown). Furthermore, phosphorylation of AKT was also stimulated after incubation of Sertoli cells in the presence of IGF-I (100 ng/ml), insulin (5 µg/ml), forskolin (10 µM), or FSH (0.9 IU/ml) for 4 h (data not shown).



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  		Figure 4. Time course of FSH effects on phosphorylation of AKT in immature rat Sertoli cells. The cells were treated with FSH (0.9 IU/ml) for 10, 30, 60, and 240 min, and the effects on p-AKT were determined. The cells treated with IGF-I (100 ng/ml) and IGF-I plus FSH for 10 min were included as positive controls. Further details are given in Fig. 2Go.

 
To investigate whether the synergistic effects of FSH and cAMP on AKT phosphorylation were mediated via the activation PI3K, we investigated the effects of these treatments in the presence and absence of LY294002, a specific inhibitor of PI3K. Sertoli cells were preincubated with LY294002 (10 µM) for 30 min, followed by incubation with FSH or cAMP for 10 min. The cells were then treated with IGF-I for 10 min or 2 h. The cells were lysed, and cellular proteins were analyzed for p-AKT and total AKT proteins by Western blotting. As shown in Fig. 5AGo, short-term treatment with FSH alone did not have any significant effect on the levels of p-AKT. IGF-I caused a significant increase in p-AKT levels, and this effect was amplified in the presence of FSH or cAMP. Incubation with the PI3K inhibitor caused a significant inhibition of the stimulatory effects of all agonists on AKT phosphorylation. In the cells that were treated with different agonists for 2 h, both FSH and cAMP analog caused a significant stimulation of p-AKT when added alone. FSH and cAMP analog also enhanced the effects of IGF-I on p-AKT as described above. These effects of FSH and cAMP analog on basal and IGF-I-stimulated p-AKT were also significantly inhibited by preincubation with the PI3K inhibitor (Fig. 5BGo).



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  		Figure 5. Effects of PI3K inhibitor on phosphorylation of AKT in Sertoli cells. The cells were preincubated in the presence or absence of LY294002 (10 µM) for 30 min, followed by treatment with FSH (F; 0.9 IU/ml) or 8-bromo-cAMP (A; 40 µM) for 10 min. The cells were then incubated in the presence or absence of IGF-I (100 ng/ml) for 10 min (A) or 2 h (B). The cell lysates were analyzed for p-AKT and total AKT by Western blotting. Further details are given in Fig. 2Go.

 
Previous studies have shown that FSH induces the secretion of IGF-I in immature Sertoli cells, but inhibits the secretion of IGF-BP3 (7, 10, 15, 16, 27). To confirm the findings of the previous studies, we determined the levels of IGF-I and IGF-BPs in Sertoli cell-conditioned medium after treatment with FSH for 4 h. As expected FSH caused an increase (131% of the controls) in the secretion of IGF-I and a decrease (72% of controls) in the secretion of IGF-BP3. The effects of forskolin and cAMP analog were more marked on IGF-I secretion (171% and 168% of controls, respectively) and IGF-BP3 levels (31% and 69% of the controls, respectively) after 24 h of treatment. Therefore, we assumed that the effects of FSH and cAMP analogs on AKT phosphorylation in Sertoli cells could be due to either increased secretion of IGF-I or decreased secretion of IGF-BP3. To explore this possibility, the effects of IGF-I, FSH, and 8-bromo-cAMP on AKT phosphorylation were studied in the presence and absence of IGF-BP3. The cells were preincubated with IGF-BP3 (5 µg/ml) for 30 min, followed by treatment with IGF-I, FSH, and cAMP analog for 10 min or 2 h as described above. As shown in Fig. 6Go, both short-term (10 min) and long-term (2 h) effects of all agonists were completely blocked by pretreatment with IGF-BP3. In addition, the levels of AKT protein were significantly reduced in Sertoli cells after treatment with IGF-BP for 2 h.



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  		Figure 6. Effects of IGF-BP3 on phosphorylation of AKT in Sertoli cells. The cells were preincubated with human recombinant IGF-BP3 (5 µg/ml), followed by treatment with FSH and 8-bromo-cAMP for 10 min, then incubated in the presence or absence of IGF-I for 10 min (A) and 2 h (B). The cell lysates were analyzed for p-AKT and total AKT by Western blotting. Further details are given in Figs. 2Go and 5Go.

 
We studied the effects of PI3K inhibitor and IGF-BP3 on morphology and cell survival to explore the possibility that inhibition of the effects of IGF-I might influence the survival of immature Sertoli cells in culture. The cells were treated with LY294002 (10 µM) or IGF-BP3 (5 µg/ml) for 24 h and were analyzed by phase contrast microscopy and after fluorescent staining of nuclei. Treatment with the PI3K inhibitor and IGF-BP3 did not have any significant effect on cell morphology (Fig. 7Go) or survival, as no significant differences in the presence of pycnotic nuclei or nuclear fragmentation (<5% of the total cells) were observed in cell cultures after treatment with either of the agents for 24 h. A summary of three experiments is shown in Fig. 8AGo. Furthermore, as shown in Fig. 8BGo, incubation of cells with IGF-BP3 or LY294002 for 24 h did not result in the formation of oligonucleosomes. In addition, total protein and DNA content in untreated cells and after treatment with IGF-BP3 or LY294002 remained unchanged (243.3 ± 0.64, 203.3 ± 34.5, and 207.5 ± 4.4 µg protein/well, respectively; 7.67 ± 2.0, 10.03 ± 1.6, and 8.15 ± 1.6 µg DNA/well, respectively). Furthermore, the number of cells attached to the coverslips was not altered by these treatments (data not shown). There were, however, subtle differences in the morphology of Sertoli cells treated with IGF-BP3. These cells exhibited an increased degree of aggregation compared with untreated cells.



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  		Figure 7. Effects of PI3K inhibitor and IGF-BP3 on morphology and survival of immature rat Sertoli cells. The cells were cultured in the presence or absence of LY29002 (10 µM) or IGF-BP3 (5 µg/ml) for 24 h. The cells were fixed in 4% paraformaldehyde, and the nuclei were stained with DAPI. The morphology of the cells was observed by phase contrast microscopy, and DAPI staining was determined by fluorescence microscopy.

 


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  		Figure 8. Effects of PI3K inhibitor and IGF-BP3 on nuclear condensation and oligonucleosome formation in immature rat Sertoli cells. The cells were left untreated (Control) or were treated with IGF-BP (5 µg/ml) or LY29002 (10 µM) for 24 h. A, The morphology of the cells was observed by phase contrast microscopy, and DAPI staining was determined by fluorescence microscopy. Results from three separate experiments are shown as a percentage of viable cells with normal nuclear morphology. B, Total genomic DNA (10 µg/lane) was electrophoresed in 2% agarose gel and stained with ethidium bromide along with DNA molecular weight markers (0.5–10 kb).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the present study demonstrate that IGF-I rapidly and potently activates the PI3K/AKT signaling pathway in rat Sertoli cells. Of considerable interest are our observations demonstrating that the endocrine factor FSH dramatically enhances IGF-I-dependent AKT phosphorylation. The effects of FSH on AKT/PKB signaling are mediated by cAMP, but appear to be independent of activation of PKA. Furthermore, FSH effects are mediated by PI3K signaling and may involve increased secretion of endogenous IGF-I or inhibition of IGF-BPs in immature rat Sertoli cells. These results clearly demonstrate that gonadotropins are capable of amplifying the intracellular signals initiated by IGF-I in Sertoli cells.

Several studies have previously established that Sertoli cells secrete IGF-I, and the secretion of IGF-I is stimulated by FSH. In vivo synthesis of IGF-I is restricted to Sertoli cells in immature animals when these cells are most responsive to FSH (45). FSH can also increase the secretion of IGF-I by rat Sertoli cells after removal of endogenous FSH by hypophysectomy and GnRH antagonist treatment (7, 8, 14, 18, 27, 46, 47). In addition, FSH inhibits the secretion of IGF-BP3 from Sertoli cells (14, 15, 16, 48). It is, therefore, commonly believed that the effects of FSH on the proliferation and differentiation of immature Sertoli cells in vivo are mediated at least in part by IGF-I (2, 28, 29, 49, 50, 51, 52). This idea is supported by multiple effects of IGF-I on in vitro proliferation and function of Sertoli cells, which include stimulation of DNA synthesis; lactate production; secretion of transferrin, plasminogen activator, and inhibin; and glucose transport (4, 20, 21, 22, 23, 24, 25). Which of these biological effects of IGF-I on Sertoli cells are dependent on the activation of the PI3K/AKT signaling pathway remains to be established.

IGF-I inhibits FSH-dependent aromatase activity in immature rat Sertoli cells (26), a biological response to FSH that gradually decreases with increasing differentiation of Sertoli cells (2, 38, 53). Our results on the effects of IGF-I on DNA synthesis and aromatase activity in immature rat Sertoli cells confirm the findings of earlier studies. The paradoxical effects of IGF-I on proliferation and aromatase activity in Sertoli cells are not unique to this growth factor. Other paracrine and autocrine factors that stimulate the proliferation and/or functions of Sertoli cells, such as epidermal growth factor, PmodS, TNF{alpha}, and IL-1, inhibit FSH-dependent aromatase activity in immature Sertoli cells (39, 54, 55, 56).

In the present studies acute treatment with FSH caused a synergistic increase in IGF-I-mediated AKT phosphorylation. When added alone for 10 min, FSH had no effect on phosphorylation of AKT; however, it significantly amplified the effects of IGF-I. Increased levels of intracellular cAMP appear to mediate the effects of FSH on IGF-I-stimulated activation of AKT, because treatment with forskolin and cAMP analogs exerted identical effects. However, the stimulatory actions do not seem to involve cAMP-dependent protein kinase (PKA) based on the inability of H89 to interfere with IGF-I- and FSH-induced AKT phosphorylation. PKA-dependent and independent effects of cAMP have been reported in previous studies (57, 58), and a similar lack of PKA involvement in FSH/cAMP effects on AKT phosphorylation has previously been established in rat granulosa cells (37).

The inhibition of synergistic effects of FSH and IGF-I analog on AKT phosphorylation by the PI3K inhibitor (LY294002) in the present study and in rat granulosa cells (37) indicates that FSH enhances the activity of PI3K. As the stimulatory effect of FSH was completely blocked by IGF-BP3, it is logical to assume that the FSH effects on PI3K are dependent on IGF-I signaling. Hence, this effect is presumably exerted at an early step in the signal transduction pathway, such as the phosphorylation of insulin receptor substrate proteins that recruit and allow for the activation of PI3K (31). Alternatively, FSH could regulate the activity of phosphotyrosine phosphatases, which would maintain or elevate the level of tyrosine-phosphorylated insulin receptor substrate proteins (59). FSH may also regulate the activity of small G proteins that could possibly alter the activity of p21ras, which, in turn, could regulate the catalytic activity of PI3K (37, 60, 61). Although the exact mechanism by which FSH amplifies IGF-I signaling is unknown, this certainly opens many questions that deserve further exploration.

In addition to its synergistic effects on IGF-I-stimulated AKT phosphorylation, FSH caused a significant increase in AKT phosphorylation when added alone for longer times. These effects of FSH on AKT phosphorylation also seem to require increased intracellular levels of cAMP. Similar cAMP-dependent stimulation of AKT phosphorylation has previously been reported in response to FSH in rat granulosa cells (37). Preincubation with the PI3K inhibitor (LY294002) and IGF-BP3 inhibited these effects of FSH and cAMP. FSH is known to stimulate the secretion of IGF-I and inhibit the secretion of IGF-BP3 in immature rat Sertoli cells (9, 15, 16). The results of the present study also show that FSH and cAMP analogs stimulate the secretion of IGF-I and inhibit the secretion of IGF-BP3 in immature rat Sertoli cells. Therefore, the effects of FSH on AKT phosphorylation in longer incubations could be due to a combination of increased secretion of endogenous IGF-I, decreased IGF-BP3, and the maintenance of the synergistic action of FSH on IGF-I-dependent PI3K activation.

On the basis of the results presented here, it is concluded that FSH not only stimulates the secretion of IGF-I by rat Sertoli cells, it also enhances its effects on PI3K/AKT signaling. These effects of FSH on IGF-I signaling are mediated by increased intracellular levels of cAMP, but appear to be independent of PKA. At present, the exact role of AKT signaling in the specific effects of IGF-I on Sertoli cells is not known. Unlike granulosa cells, the inhibition of PI3K and endogenous IGF-I action (by IGF-BP3) did not influence the survival of Sertoli cells over a period of 24 h (35). Further studies are needed to establish the significance of individual signaling pathways in specific functions of IGF-I in Sertoli cells and to determine the role of FSH in these processes.


   Acknowledgments
 
The authors are grateful to Cecille Millena for technical assistance, Susan Chavez for performing E2 RIAs, and Dr. Curt Pfarr for his assistance in performing morphological analysis of the cells.


   Footnotes
 
This work was supported by NIEHS Grant ES-10232 (to S.A.K.) and NIH Grant 38813, V.A., and USDA (to J.S.D.).

Abbreviations: CREB, cAMP response element-binding protein; DAPI, 4',6-diamidino-2 phenylindole; IGF-BP, IGF-binding protein; p-AKT, phosphorylated form of AKT; PKB, protein kinase B.

Received October 12, 2001.

Accepted for publication February 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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