Abstract
Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells.
POSTNATAL DEVELOPMENT and function of testicular Sertoli cells is regulated primarily by FSH, a glycoprotein hormone secreted by the pituitary gland (1, 2). In the prepubertal testis, FSH is required for proliferation of Sertoli cells to achieve the adult number of Sertoli cells (3). This proliferative stage of Sertoli cell development is also characterized by the presence of FSH-dependent cytochrome P450 aromatase activity in neonatal and prepubertal Sertoli cells (4–6). During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development (7, 8). Overexpression of the aromatase gene (Cyp19a1) results in testicular hyperplasia (9), whereas male mice deficient in aromatase [aromatase knockout (ArKO)] are infertile (10). The proliferative period of rat Sertoli cells ends around 15 d of age and is followed by differentiation of Sertoli cells into mature adult-type Sertoli cells (4). During this later stage of development, the response of the Sertoli cells to FSH in terms of proliferation and aromatase activity is significantly reduced (11, 12). The nature of the intracellular signaling mechanisms in Sertoli cells that are involved in the differential effects of FSH during different stages of development remains unknown.
The presence or absence of growth factors and cytokines in the testis and their interaction with FSH signaling pathways may be instrumental in the differential responses of Sertoli cells to FSH during specific developmental stages (13–15). Epidermal growth factor (EGF) has been shown to be critical for spermatogenesis in male mice (16). Removal of submandibulary glands in mice leads to disruption of spermatogenesis, which can be reversed by exogenous EGF (16). It is not known whether these effects on spermatogenesis are a consequence of direct effects of EGF on germ cells or through its effects on Sertoli cells. Several studies have shown the presence of EGF receptors (EGFRs) in testicular cells in various species (17–21). EGF and a related ligand, TGFα, which binds to the EGFR and exerts biological effects nearly identical to those of EGF on target cells, are present in the postnatal testis and regulate the proliferation and differentiation of Sertoli cells (20, 22–24). Other evidence indicates that EGF treatment can modulate the effects of FSH on Sertoli cell development and function. Whereas EGF potentiates the effects of FSH on lactate and transferrin production and stimulates proliferation in immature Sertoli cells, EGF also inhibits FSH effects on aromatase activity (25, 26). The reduction in aromatase activity is indicative of the termination of the proliferative period and the initiation of functional differentiation of Sertoli cells (4). The intracellular signal pathways involved in the inhibitory actions of EGF on Sertoli cells are unknown.
The present study investigates the intracellular events that take place downstream of FSH and EGF receptors and examines which intracellular signals may be involved in their effects on aromatase activity in immature rat Sertoli cells. Whereas it is generally appreciated that FSH stimulates cAMP/protein kinase A (PKA) signaling in Sertoli cells (12, 27), additional signaling cascades downstream of cAMP have been identified, including the activation of phosphatidylinositol 3-kinase (PI3K)/AKT1 (protein kinase B) signaling pathway (28–30) and the p42 ERK2 and p44 ERK1 MAPK signaling pathway (also named MAPK1 and MAPK3, respectively) (13).
The involvement of these intracellular signaling mechanisms in the effects of FSH on aromatase expression in immature rat Sertoli cells is unknown. However, recent studies performed in rat granulosa cells demonstrated that the PI3K/AKT1 pathway may be important for estradiol production (31). The current experiments were designed to investigate the hypothesis that FSH-induced PI3K/AKT signaling plays an important role in aromatase activity in immature rat Sertoli cells. Furthermore, we sought to determine whether the PI3K/AKT1 or ERK signaling pathways mediate the actions of EGF in immature Sertoli cells because EGF plays an inhibitory role in FSH-stimulated aromatase activity. Our results indicate that the stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production are dependent on an active PI3K/AKT1 signaling pathway. Furthermore, our experiments demonstrate that EGF inhibits the expression of aromatase mRNA and protein in response to FSH, and that these inhibitory effects are critically dependent on the activation of the ERK-MAPK signaling pathway.
RESULTS
Effects of FSH on Intracellular Signaling and Aromatase Expression
Preliminary experiments indicated that, after treatment with FSH, increased levels of aromatase mRNA were observed within 1 h, maximal increases were observed within 8 h of treatment, and levels of aromatase mRNA remained elevated for longer than 24 h (data not shown). Because preliminary results indicated that the changes in aromatase mRNA occur rapidly, we selected incubations times up to 8 h for our studies on aromatase mRNA and protein levels. Shown in Fig. 1, A and B, are representative time courses for aromatase mRNA and protein after treatment with control media or FSH for up to 6 h. Aromatase mRNA and protein levels were very low in Sertoli cells treated with control media. Increased levels of aromatase mRNA and protein were observed after treatment with FSH for 2–6 h. Levels of L-19 mRNA and β-actin protein were unchanged by FSH treatment. As anticipated, treatment with FSH (1 IU/ml) for 24 h resulted in a significant increase (50-fold; P < 0.01, n = 5) in estradiol accumulation in primary cultures of rat Sertoli cells.
FSH Stimulates Aromatase mRNA and Protein in Immature Rat Sertoli Cells Rat Sertoli cells were treated for up to 8 h with FSH (1 IU/ml). A, Total RNA was isolated and semiquantitative RT-PCR was performed as described in Materials and Methods. Shown are levels of aromatase and L-19 transcripts in a representative experiment. B, Cellular protein extracts were prepared and Western blot analysis was performed as described in Materials and Methods. Shown are levels of aromatase and β-actin proteins in a representative experiment. C, Preparation of cellular protein extracts and Western blot analysis were performed as described in Materials and Methods. Shown are levels of aromatase, phospho-AKT, phospho-ERK, and β-actin proteins in a representative experiment.
FSH is known to induce its cellular responses via activation of multiple intracellular signaling pathways (28–30, 32, 33). Experiments were performed to determine whether increases in PI3K/AKT and ERK signaling events occurred during the induction of aromatase in FSH-treated rat Sertoli cells. The activation of ERK and AKT were determined by Western blot analysis using activation state-specific antibodies that recognize the phosphorylated forms of these enzymes. Figure 1C shows a representative Western blot demonstrating that increases in the phosphorylation of AKT and ERK occurred within 1 h of treatment with FSH; whereas substantial increases in aromatase protein accumulation occurred after 2 h of treatment with FSH.
To determine the possible role of each intracellular signaling pathway, rat Sertoli cells were pretreated with well-characterized inhibitors of the PI3K/AKT and ERK signaling pathways. We observed that pretreatment with the PI3K inhibitor LY294002 reduced the stimulatory affect of FSH on aromatase mRNA levels (Fig. 2A). Furthermore, we observed that pretreatment with LY294002 also prevented the FSH-induced increase in aromatase protein (Fig. 2B). The levels of phosphorylated AKT proteins were evaluated in these extracts to verify the effectiveness of LY294002. We observed that pretreatment with LY294002 completely inhibited the stimulatory effect of FSH on the phosphorylation of AKT. We also observed that inhibition of PI3K/AKT signaling was associated with a significant inhibition of FSH-stimulated estradiol accumulation (Fig. 2C). Pretreatment with LY294002 resulted in a 70% reduction (P < 0.05, n = 3) in estradiol production after treatment with FSH.
The Phosphatidylinositol-3-Kinase Inhibitor LY294002 Inhibits FSH-Stimulated Increases in Aromatase mRNA, Aromatase Protein, and Estradiol Synthesis in Immature Rat Sertoli Cells Rat Sertoli cells were pretreated for 30 min with LY294002 (20 μm) before treatment with FSH (1 IU/ml). A, Shown are levels of aromatase and L-19 transcripts in a representative experiment. B, Shown are levels of aromatase and β-actin proteins in a representative experiment. C, Levels of estradiol were determined in conditioned media after 24 h of incubation in the presence or absence of FSH. *, P < 0.05, compared with FSH alone; n = 3. DMSO, Dimethylsulfoxide.
In contrast to the inhibitory effects exerted by the PI3K inhibitor, treatment with the MAPK kinase 1 (MEK-1) inhibitor PD98059 resulted in an enhancement of FSH-induced aromatase mRNA levels (Fig. 3A). Likewise, treatment with PD98059 resulted in an increase in aromatase protein levels after treatment with FSH (Fig. 3B). The effectiveness of PD98059 was verified by its ability to inhibit the phosphorylation of ERK proteins but not AKT in response to treatment with FSH. In keeping with the stimulatory affects of PD98059 on FSH-induced aromatase mRNA and protein expression, we observed that pretreatment with PD98059 significantly elevated (81%; P < 0.05, n = 3) estradiol production in response to FSH treatment (Fig. 3C).
Treatment with the MEK-1 Inhibitor PD98059 Elevates Aromatase mRNA, Aromatase Protein, and Estradiol Synthesis in FSH-Stimulated Immature Rat Sertoli Cells Rat Sertoli cells were pretreated for 60 min with PD98059 (25 μm) before treatment with FSH (1 IU/ml). A, Shown are levels of aromatase and L-19 transcripts in a representative experiment. B, Shown are levels of aromatase, phospho-AKT, phospho-ERK, and β-actin proteins in a representative experiment. C, Levels of estradiol were determined in conditioned media after 24 h of incubation in the presence or absence of FSH. *, P < 0.05 compared with FSH alone; n = 3. DMSO, Dimethylsulfoxide.
Effects of EGF on Estradiol Secretion and Aromatase Expression
Our first set of experiments determined the effect of EGF on basal and FSH-stimulated estradiol production and the induction of aromatase mRNA and protein (Fig. 4). Treatment with EGF (10 ng/ml) reduced basal levels of estradiol compared with controls (31%; P < 0.05, n =3) and FSH-stimulated estradiol secretion by 80% (P < 0.05; n = 3) (Fig. 4A). Aromatase mRNA levels were very low in control and EGF-treated cells. The reduction in FSH-stimulated estradiol production in cells treated with EGF was correlated with a reduction in FSH-stimulated aromatase mRNA levels in rat Sertoli cells (Fig. 4B). Treatment with increasing concentrations (0.1 to 10 ng/ml) of EGF resulted in concentration-dependent reductions in FSH-stimulated aromatase mRNA (Fig. 4B). As observed for estradiol and aromatase mRNA, the FSH-induced increase in aromatase protein levels was also reduced in the presence of EGF (Fig. 4C). Taken together, these results demonstrate that EGF effectively reduced the levels of aromatase protein and mRNA, as well as the production of estradiol in FSH-stimulated rat Sertoli cells.
Treatment with EGF Inhibits FSH-Stimulated Estradiol Accumulation, Aromatase mRNA, and Aromatase Protein in Immature Rat Sertoli Cells Rat Sertoli cells were treated with various concentrations of EGF or a combination of FSH plus EGF. A, Estradiol levels were determined in conditioned media after 24 h treatment with either control media, FSH (1 IU/ml), EGF (10 ng/ml), or EGF plus FSH as indicated in Materials and Methods. B, Levels of aromatase and L-19 transcripts in a representative experiment were determined after 4-h treatments with control media or FSH (1 IU/ml) in the presence of increasing amounts of EGF (0–10 ng/ml). C, Aromatase protein was determined after 4-h incubations with either control media (CTL), EGF (10 ng/ml), FSH (1 IU/ml), or the combination of FSH plus EGF. Shown are levels of aromatase and β-actin proteins in a representative experiment. *, P < 0.05 compared with FSH alone; n = 3.
Effects of EGF on Intracellular Signaling in Rat Sertoli Cells
EGF (34, 35) is known to induce pleiotropic responses in many cell types via activation of multiple intracellular signaling pathways. Recent data have implicated the MAPK signaling pathway in the regulation of steroid synthesis (36, 37). Experiments were performed to determine whether EGF elicits common signaling events that may explain its inhibitory effects on FSH-stimulated estradiol production. The time course and concentration response to EGF treatment of rat Sertoli cells are shown in Fig. 5. EGF stimulated a rapid elevation of levels of phosphorylated ERK and AKT proteins. The effects of EGF on ERK and AKT phosphorylation were maximal at 5 and 15 min of incubation (Fig. 5A). In longer incubations, the stimulatory effects of EGF on ERK phosphorylation were apparent, but reduced levels of these phosphorylated proteins were observed. Concentration-response studies with EGF treatment for 5 min showed that activation of ERK and AKT signaling was maximal at 10–25 ng/ml EGF (Fig. 5B).
EGF Stimulates the PI3K/AKT and ERK Signaling in Immature Rat Sertoli Cells A, Rat Sertoli cells were treated for up to 120 min with EGF (10 ng/ml). B, Rat Sertoli cells were treated with increasing concentrations of EGF (1–50 ng/ml) for 5 min. Shown are levels of phospho-AKT, phospho-ERK, pan-ERK aromatase, and β-actin proteins in a representative experiment.
To further examine the possible role of EGFR signaling in the inhibition of aromatase, Sertoli cells were treated in the presence or absence of the selective EGFR tyrosine kinase inhibitor AG1478 (Fig. 6). Treatment with AG1478 (100 nm) did not alter basal levels of aromatase mRNA or protein. However, pretreatment with AG1478 abrogated the inhibitory effects of EGF on aromatase mRNA (Fig. 6A) and protein (Fig. 6B) in FSH-treated Sertoli cells. Western blot analysis was performed to verify the effectiveness of EGFR signaling blockade in response to AG1478 treatment. In cells treated with vehicle, the stimulatory effects of EGF on ERK phosphorylation were much greater than the stimulatory effects of FSH on ERK phosphorylation in Sertoli cells. Pretreatment with AG1478 completely blocked the phosphorylation of ERK and AKT observed in response to EGF (Fig. 6C). Pretreatment with AG1478 also inhibited FSH-induced ERK signaling. However, AG1478 only partially reduced the stimulatory effect of FSH on AKT phosphorylation.
Treatment with the EGFR Tyrosine Kinase Inhibitor AG1478 Prevents EGFR-Mediated Signaling Events and Prevents the Inhibitory Affect of EGF on FSH-Stimulated Aromatase Expression Rat Sertoli cells were incubated for 60 min with AG1478 (100 nm) before treatment with control media (CTL), FSH (1 IU/ml), or FSH plus EGF (10 ng/ml) for 4 h. A, Shown are levels of aromatase and L-19 transcripts in a representative experiment. B, Shown are levels of aromatase and β-actin proteins in a representative experiment. C, Rat Sertoli cells were preincubated with AG1478 (100 nm) for 60 min followed by a 15-min treatment with control media (CTL), FSH (1 IU/ml), EGF (10 ng/ml), or EGF plus FSH. Shown is the Western blot analysis of signaling proteins (phospho-AKT and phospho-ERK) and β-actin in a representative experiment. DMSO, Dimethylsulfoxide.
Effects of the MEK-1 Inhibitor PD98059 and the PI3K Inhibitor LY294002 on the Inhibitory Response to EGF
To determine the possible role of ERK signaling in the inhibitory responses to EGF, rat Sertoli cells were treated with the MEK-1 inhibitor, PD98059 (50 μm) to interrupt ERK activation. Figure 7A shows that treatment with FSH increased estradiol production and that concomitant treatment with the MEK-1 inhibitor PD98059 (50 μm) augmented the stimulatory response to FSH. As observed earlier, treatment with EGF inhibited the stimulatory effect of FSH on estradiol production. Pretreatment with PD98059 (50 μm) for 60 min before the addition of FSH and EGF abrogated the inhibitory affect of EGF on estradiol production in FSH-treated Sertoli cells (Fig. 7A). In contrast, pretreatment with the PI3K inhibitor, LY294002 (20 μm), did not reverse the inhibitory effects of EGF. In fact, it inhibited estradiol production in FSH-treated Sertoli cells, as shown previously in Fig. 2C (data not shown).
Treatment with the MEK-1 Inhibitor PD98059 Abrogates the Inhibitory Effects of EGF on FSH-Stimulated Estradiol Accumulation and Aromatase Expression in Rat Sertoli Cells Immature rat Sertoli cells were pretreated with PD98059 (50 μm) for 60 min before treatment with control media (C), FSH (1 IU/ml), EGF (10 ng/ml), or EGF plus FSH. A, Estradiol levels were determined in conditioned media after 24 h of incubation as indicated in Materials and Methods. B, Levels of aromatase and L-19 transcripts were determined after 4 h of treatment. C, Levels of aromatase and β-actin proteins were determined after 4 h of treatment. D, Levels of phospho-ERK and β-actin proteins were determined after 15 min of treatment. *, P < 0.05 compared with FSH alone; n = 3. DMSO, Dimethylsulfoxide.
Analysis of levels of aromatase mRNA revealed that pretreatment with PD98059 (50 μm) had little effect on basal aromatase mRNA levels but elevated the stimulatory effect of FSH. Furthermore, pretreatment with PD98059 prevented the inhibitory effects of EGF on FSH-stimulated aromatase mRNA levels (Fig. 7B). Western blot analysis of aromatase protein was performed to determine whether the changes in aromatase mRNA levels were reflected by changes in protein. As seen in Fig. 7C, pretreatment with PD98059 (50 μm) prevented the inhibitory effects of EGF on aromatase protein levels in FSH-treated Sertoli cells.
Experiments were performed to validate the effectiveness of the MEK-1 inhibitor. Figure 7D shows that treatment with EGF resulted in robust elevations of ERK phosphorylation in 30-min incubations. Pretreatment with PD98059 (50 μm) substantially reduced ERK phosphorylation in response to treatment with EGF. In contrast, pretreatment with PD98059 had only slight effects on the levels of AKT phosphorylation in response to FSH or EGF (data not shown). Taken together, the results indicate that inhibition of ERK MAPK signaling by employing the MEK-1 inhibitor PD98059 resulted in a marked reduction in the inhibitory affects of EGF on estradiol and aromatase production.
DISCUSSION
The present results confirm that FSH is capable of increasing aromatase levels and estradiol production in Sertoli cells isolated from 10-d-old rats, a developmental stage characterized by FSH-induced Sertoli cell proliferation. Although the mechanism of FSH action in Sertoli cells is well known to involve the activation of adenylyl cyclase and elevation of cAMP (12), recent studies indicate that additional signaling cascades are activated downstream of cAMP, including activation of PI3K/AKT (28–30) and ERK (13) signaling pathways. We observed that increases in the phosphorylation of AKT and ERK occurred within 1 h of treatment with FSH, a time when aromatase mRNA levels were increasing, but before substantial increases in aromatase protein accumulation. The present studies also show that each pathway uniquely regulates the effects of FSH on aromatase expression and estradiol production. An active PI3K/AKT signaling pathway was required for the stimulatory actions of FSH aromatase expression and estradiol production, whereas an active ERK/MAPK pathway inhibited estradiol production and aromatase expression. Furthermore, we show that EGF inhibited FSH-induced aromatase expression and estradiol synthesis in immature Sertoli cells by an ERK/MAPK-dependent signaling pathway.
Sertoli cells undergo a rapid phase of proliferation in the neonatal rat testes until d 15 of postnatal life. This period of proliferation is dependent on FSH and coincides with high expression of aromatase in Sertoli cells. To determine the relative contribution of the PI3K and ERK signaling pathways to estradiol production, we treated Sertoli cells isolated from 10-d-old rats with pathway-specific inhibitors. We observed that blockade of PI3K signaling strongly inhibited FSH-dependent estradiol production and FSH-stimulated aromatase mRNA and protein in Sertoli cells. The expression of the aromatase gene is regulated by cAMP/PKA signaling and FSH-responsive transcription factors such as cAMP-response element binding protein (CREB); steroidogenic factor-1 (SF-1/NR5A1), an orphan nuclear receptor; and GATA-4 and -6 binding proteins (38–40). We (28) and others (29, 30) have shown that FSH and cAMP analogs stimulate the phosphorylation of AKT in immature rat Sertoli cells, which is blocked by chemical inhibitors of PI3K. The inhibition of FSH-induced estradiol and AKT phosphorylation by the PI3K inhibitor LY294002 in the present study suggests a prominent role for the PI3K/AKT signaling pathway in the regulation of aromatase in immature rat Sertoli cells. The results cannot be explained by an inhibition of cell viability because LY294002 does not alter the survival of Sertoli cells under these primary culture conditions (28). Additional support for a role of AKT in the induction of aromatase is derived from studies in rat granulosa cells demonstrating that adenovirus-mediated overexpression of a constitutively active form of AKT (myristoylated-AKT) augmented the stimulatory effects of FSH on estradiol production, whereas, overexpression of dominant-negative AKT prevented the stimulatory effects of FSH on estradiol production (31). Furthermore, PKA activation appears to be critical for aromatase activity because the inhibition of FSH-induced estradiol production after treatment with the PKA inhibitor H-89 was not overcome by overexpression of myristoylated-AKT (31). Recent observations by Krylova et al. (41) implicate PI3K-generated phospholipids as putative ligands for the nuclear receptor steroidogenic factor 1 (SF-1). These authors demonstrated that the binding of phosphatidylinositol-derived second messengers to SF-1, especially the direct products of PI3K activation, resulted in maximal SF-1-dependent activation of an aromatase reporter in HepG2 hepatocytes. Taken together, these findings suggest that estradiol production in FSH-treated Sertoli cells may require PKA, PI3K, and AKT signals for optimal induction of the aromatase gene.
In an effort to understand the nature of intracellular mechanisms involved in the inhibition of FSH-induced estradiol production, we performed an analysis of effects of EGF on the activation of ERK and PI3K/AKT signaling in immature rat Sertoli cells. We observed that EGF stimulated a dose- and time-dependent induction of both PI3K/AKT and ERK signaling pathways in Sertoli cells. Whereas the sensitive and rapid onset of EGF signaling in the present study is comparable to observations on EGF stimulation of ERK signaling in ovarian cells (42, 43) and Sertoli cells (13), the stimulatory effect of EGF on AKT phosphorylation has, to our knowledge, not been previously reported in rat Sertoli cells. We observed that the specific EGFR kinase inhibitor AG1478 effectively blocked EGF-stimulated ERK and AKT phosphorylation in Sertoli cells, which demonstrates that the initiation of ERK and AKT signaling in Sertoli cells is initiated by EGFR tyrosine kinase activation. This leads to the activation of guanine nucleotide exchange factors (like son of sevenless protein homolog 1, SOS1) and initiation of the Ras/Raf/MEK/ERK signaling cascade (44). The activation of PI3K by EGF occurs via association of the p85 subunit of PI3K (PIK3R1) with the activated EGFR (45) or via Ras-mediated activation of the p110 catalytic subunit of PI3K (PIK3CA) (46). Our studies indicate that whereas EGF induced a robust activation of both MEK/ERK and PI3K/AKT signaling pathways in immature Sertoli cells, only the ERK pathway was involved in the inhibitory effects of EGF on estradiol synthesis and aromatase expression. This assumption is supported by the data demonstrating the reversal of the inhibitory effects of EGF by pretreatment with the EGFR tyrosine kinase inhibitor AG1478, or by pretreatment with the MEK-1 inhibitor PD98059, whereas pretreatment with the PI3K inhibitor did not alter the inhibitory activity of EGF. Furthermore, inhibition of the MEK/ERK pathway resulted in increases in FSH-induced expression of aromatase in Sertoli cells, indicating that ERK activity may provide a tonic inhibitory effect on aromatase expression. These results are in agreement with a previous study showing that overexpression of a constitutively active MEK protein in rat granulosa cells inhibited FSH-induced estradiol production (31). These results are also consistent with studies showing inhibitory effects of the ERK/MAPK pathway on progesterone synthesis in ovarian cells (47, 48) and testosterone synthesis in Leydig cells (37). The present studies extend these reports by providing evidence that the changes observed in estradiol production are associated with changes in aromatase mRNA and aromatase protein levels in immature Sertoli cells. In other studies (our unpublished data), EGF did not influence activity of the aromatase enzyme after its induction by FSH (as measured by estradiol production), suggesting that the inhibitory effects of EGF are primarily exerted at the level of aromatase gene expression.
The intracellular mechanisms downstream of ERK signaling that contribute to the inhibition of aromatase gene expression are unknown. Aromatase gene expression is known to be positively regulated by cAMP response element-binding protein, SF-1, and GATA (49), whereas other factors serve as corepressors, e.g. dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome (Dax-1, NR0B1) and Wilm’s tumor suppressor gene (40, 49–52). Studies by Hammer and Ingraham (50) and Hammer et al. (51) indicate that maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on ERK-dependent phosphorylation of a single serine residue (Ser-203) located in a major activation domain of the protein. It is unknown whether FSH- or EGF-induced ERK signaling results in the phosphorylation of SF-1 in Sertoli cells; however, this does not appear to play a stimulatory role in the expression of aromatase in response to FSH because inhibition of MEK/ERK signaling did not dampen FSH-induced aromatase expression. In fact, the opposite occurred: inhibition of ERK signaling elevated FSH-aromatase mRNA and protein and estradiol production. It seems more likely that the ERK-dependent phosphorylation site on SF-1 contributes to the recruitment of other regulatory proteins such as nuclear receptor corepressor 2 (NCOR2; also known as SMRT, silencing mediator for retinoid and thyroid receptors), which represses the transcriptional activity of SF-1 (51). Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome gene 1) is another Sertoli cell factor that represses transcription of aromatase by binding to and inhibiting SF-1. In fact, targeted disruption of Dax-1 up-regulates aromatase expression in the testis (49). Furthermore, treatment of human granulosa cells with MEK1 inhibitors reduced cellular Dax-1 levels and elevated steroidogenesis (52), suggesting that ERK signaling may regulate the levels of Dax-1 protein, which contributes to the inhibition of aromatase in Sertoli cells. It is also possible that ERK-dependent signaling may regulate the synergistic actions of SF-1 with other transcription factors such as GATA-4 (53, 54). In view of these studies and our present results, it is tempting to speculate that EGF may increase levels of corepressors and/or interactions of corepressors with SF-1 by a mechanism dependent on an active ERK signaling pathway in Sertoli cells.
It is well established that FSH-induced aromatase activity decreases gradually and is absent after 21 d of postnatal life. The effect of FSH on Sertoli cells also shifts from proliferation to functional differentiation of these cells. On the basis of previous studies and the results of the present study, it is tempting to assume that these interactions among FSH and EGF and/or TGFα (55) may be involved in autocrine regulation of the transition of Sertoli cells from the proliferative phase to functional differentiation. An intact PI3K signaling pathway (30) appears to be necessary for FSH effects on the differentiated function (transferrin and lactate production) of mature rat Sertoli cells. Furthermore, the effects of FSH are not dependent on ERK signaling at this point because FSH reduces ERK MAPK signaling in the mature Sertoli cells (13, 30). These results point to the differences in signaling responses during the switching from the early proliferative phase to the differentiation phase of Sertoli cell development. Further studies are required to delineate the role of individual factors in these processes.
We conclude that an active PI3K/AKT signaling pathway is required for the stimulatory actions of FSH on aromatase expression and estradiol secretion, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells. EGF inhibits FSH-induced expression of aromatase gene in immature rat Sertoli cells, and this effect is mediated via the ERK signaling pathway. Our studies set a stage for future investigations on the role of specific signaling pathways initiated by growth factors and cytokines, which modulate the effects of FSH during different stages of Sertoli cell development and function.
MATERIALS AND METHODS
Materials and Animals
Recombinant human FSH was obtained from National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). Human recombinant EGF was purchased from R&D Systems (Minneapolis, MN). The PI3K inhibitor LY294002, a MAPK kinase (MEK-1) inhibitor PD98059, collagenase (type 1), and deoxyribonuclease were purchased from Sigma Chemical Co. (St. Louis, MO). The EGFR tyrosine kinase inhibitor (AG1478) was purchased from Calbiochem (San Diego, CA). Antibodies against the activated forms of protein kinases phospho-Akt (no. 9271L) and Erk/MAPK (no. 9101L) were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-Actin (clone AC-15, no. A5441) antibody was purchased from Sigma. Aromatase antibody was from Serotec (Oxford, UK). The pan-ERK antibody was from BD Transduction Laboratories (Lexington, KY). Antirabbit and antimouse secondary antibodies were from Cell Signaling Technology and Sigma, respectively. All incubations and cell cultures of immature Sertoli cells were performed in Eagle’s MEM with Earle’s salts and 0.1 mΜ 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 mml-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., Gaithersburg, MD). Immature (10 d old) male Wistar Crl:(W1)BR rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and maintained with the mothers under standard conditions. All experimental protocols were approved by the Institutional Animal Care and Use Committees of the Texas Tech University Health Sciences Center and Atlanta University 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 (56) as described previously (28, 57). 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. 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 from Fisher Scientific (Pittsburgh, PA), followed by filtration through BD Falcon cell strainers (nylon mesh size, 70 μm). The cells were plated in six-well culture plates (2 × 106 cells/well) or in 24-well plates (3 × 105 cells/well) and cultured at 37 C in a humidified atmosphere of 5% CO2 and 95% air in serum-free medium. After 72 h, the cells were extensively washed to remove unattached germ cells. The cells were cultured for an additional 4-d period in the serum-free medium, with medium changes after every 48 h. The cells were then treated with different concentrations of FSH and/or EGF for specific time periods to determine their effects on aromatase mRNA and protein, conversion of androgen substrate into estradiol, and signal transduction mechanisms.
Aromatase Activity
After a 7 d of culture in serum-free medium in 24-well plates, the Sertoli cells were pretreated with MEK-1 inhibitor (PD98059, 50 μm), the PI3K inhibitor (LY294002, 20 μm), or with EGFR tyrosine kinase inhibitor (AG1478; 100 nm) for 60 min, and then treated with FSH (1 IU/ml) in the presence or absence of EGF (10 ng/ml) in the presence of aromatase substrate, 19-hydroxyandrostenedione (2.5 nm). The cells were incubated for 24 h as described previously (28). The conditioned media were collected, and estradiol levels were determined using RIA reagents from Diagnostic Products (Los Angeles, CA) as described elsewhere (58).
Western Blotting
To determine the effects of FSH and EGF on immunoreactive levels of aromatase and phosphorylated signaling proteins, immature Sertoli cells (2 × 106) were cultured in six-well plates as described above. The cells were then washed and treated with various concentrations of EGF or a combination of FSH with EGF for different time periods. For some experiments, the cells were preincubated for 60 min in the presence of a MEK-1 inhibitor (PD98059, 50 μm), a PI3K inhibitor (LY294002, 20 μm), or an EGFR tyrosine kinase inhibitor (AG1478, 100 nm) before the addition of EGF or FSH. At the end of the treatment period, the cells were washed with 1× 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 β-glycerophosphate, 1 mm Na3VO4, and 1 μg/ml leupeptin] from Cell Signaling Technology. The lysis buffer was supplemented with 1× protease inhibitor cocktail (Calbiochem, San Diego, CA) 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 content in cell lysates was determined using a DC protein assay kit from Bio-Rad Laboratories, Inc. (Richmond, CA). The cell lysates (20–40 μg protein) were subjected to 10% SDS-PAGE and then electrically (75 V, 2 h) transferred to polyvinylidene difluoride membranes from Millipore Corp (Bedford, MA). Membranes were then subjected to Ponceau staining from Sigma to confirm equivalent loading. The membranes were blocked for 60 min at room temperature in 50 mm Tris (pH 7.5), containing 0.15 m NaCl, 1 mm EDTA, 0.05% Nonidet P-40, and 5% nonfat milk. The blots were then incubated with specific antibodies that cross-react with phosphorylated forms of ERK/MAPK (Thr202/Tyr204; 1:15,000) or phospho-Akt (Ser473; 1:2000); pan-ERK (1:1,000); cytochrome P450 aromatase (1:500); and β-Actin (1:20,000). Secondary antibodies used were at 1:20,000). Blots were then developed in ECL Plus mixture from Amersham Pharmacia Biotech (Piscataway, NJ) for 1–10 min and exposed using a KODAK Image Station 440 System (Eastman Kodak, Rochester, NY).
RNA Isolation, cDNA Synthesis, and RT-PCR
Total RNA was isolated from Sertoli cells using TRIzol from Invitrogen (Carlsbad, CA) followed by chloroform extraction and isopropanol precipitation. Total RNA (2 μg) was reverse transcribed at 37 C for 1 h in a reaction mixture containing 0.5 mm deoxynucleotide triphosphate from Fisher Scientific, 400 U of mouse mammary leukemia virus reverse transcriptase from Promega (Madison, WI), 1× reverse transcriptase (RT) buffer, 0.5 mm dithiothreitol from Bio-Rad Laboratories, Inc., and 0.5 μg of oligo dT from Promega. After incubation, the RT enzyme was heat inactivated at 70 C for 5 min and the reaction cooled to 4 C. RT reactions (2 μl) were added to separate PCR mixtures (total 10 μl) containing 0.1 mm deoxynucleotide triphosphates, from Fisher Scientific (Denver, CO), 0.5 U Taq DNA polymerase from PGC Scientifics (Frederick, MD), 1× PCR buffer with 3 mm MgCl2 from Idaho Technology, Inc. (Salt Lake City, UT), and 25 pmol of the specific primers for the aromatase gene. The primer pair for aromatase was: sense, 5′-GCT TCT CAT CGC AGA GTA TCC GG-3′; and antisense, 5′-CAA GGG TAA ATT CAT TGG GCT TGG-3′. The expected molecular size was 289 bp. L-19 (a ribosomal protein) primer pair was used as internal control. The primer pair for L-19 was: sense, 5′-GAA ATC GCC AAT GCC AAC TC-3′; and antisense, 5′-TCT TAG ACC TGC GAG CCT CA-3′. The expected molecular size was 405 bp.
The following protocol was used in the Rapid Cycler (Idaho Technology): 94 C for 15 sec (initial denaturation), followed by 94 C for 0 sec, 60 C for 0 sec, 72 C for 20 sec for 35 cycles, and 72 C for 30 sec (final extension) as previously described (37). This protocol amplified products in the linear portion of the cycle curve. The PCR products were visualized on 1–2% agarose gel electrophoresis stained with ethidium bromide, using a DNA molecular mass marker of 100 bp from Promega.
Statistical Procedures
All experiments were performed at least three times using a different cell preparation with identical results. The data are presented as the means and standard deviations of the averages from multiple experiments or individual representative experiments. The differences in means were analyzed by ANOVA and Student’s t test.
Acknowledgments
This work was supported by the Veterans Affairs, Olson Center for Women’s Health, and National Institutes of Health Grants HD38813 and RCMI G12-RR03062.
Abbreviations
- Dax-1
Dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome gene 1;
- EGF
epidermal growth factor;
- EGFR
EGF receptor;
- MEK
MAPK kinase;
- PI3K
phosphatidylinositol 3-kinase;
- PKA
protein kinase A;
- RT
reverse transcriptase;
- SF-1
steroidogenic factor 1.
