Abstract
In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1–5 mm) and large (8–22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150–600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P<0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P<0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P<0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P>0.10). Treatment with 600 ng/ml GDF-9 increased (P<0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P<0.05) progesterone production (by 28%), increased (P<0.05) cell numbers (by 60%), and had no effect (P>0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13–48%) and estradiol (by 20–51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P<0.05) steroidogenesis by 27–42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.
Introduction
During ovarian follicular development, granulosa cell proliferation and differentiation are influenced by the gonadotropins and different intraovarian factors secreted by both the oocyte and the surrounding somatic cells (McGee & Hsueh 2000, Hunter et al. 2004, Spicer 2004, Juengel & McNatty 2005). Among the intraovarian factors are members of the transforming growth factor (TGF)-β superfamily, including TGF-β, activins, bone morphogenetic proteins (BMPs), and growth differentiation factors (GDFs) (Shimasaki et al. 1999, Knight & Glister 2003, Mazerbourg & Hsueh 2003, Juengel & McNatty 2005). Expression of two of these TGF-β family members, GDF-9 and BMP-15, is confined to the oocyte of primary and larger follicles in cattle (Sendai et al. 2001, Lonergan et al. 2003, Pennetier et al. 2004), sheep (Bodensteiner et al. 2000, Juengel et al. 2002), pigs (Prochazka et al. 2004), and rodents (McGrath et al. 1995, Jaatinen et al. 1999), whereas other BMPs such as BMP-2 and -4 are expressed in granulosa and/or theca cells but not the oocyte (Erickson & Shimasaki 2003, Glister et al. 2004, Fatehi et al. 2005). Studies using mice (Dong et al. 1996, Carabatsos et al. 1998) and sheep (Hanrahan et al. 2004) with inactivating mutations of the GDF-9 gene have revealed an important role of this oocyte factor in the stimulation of early follicular growth and fertility. However, little is known about the role of GDF-9 and BMPs in regulating granulosa cell function in single-ovulating species such as cattle and humans. Previous studies have revealed direct effects of GDF-9 on granulosa cell functions in rats (Vitt et al. 2002, Kaivo-Oja et al. 2003, Roh et al. 2003). Because insulin-like growth factor-I (IGF-I) is a major trophic hormone involved in follicular development (Mazerbourg et al. 2003, Spicer 2004, Hunter et al. 2004), our first objective was to determine the effects of GDF-9 and BMP-4 on IGF-I-induced cell proliferation and steroidogenesis in cultured bovine granulosa cells.
GDF-9 is most closely related to GDF-9B/BMP-15, both having homology closer to BMPs than to the activin and TGF-β proteins (Newfeld et al. 1999, Vitt et al. 2002). Members of the TGF-β family initiate signaling by assembling type I and type II serine/threonine kinase receptor complexes that activate one or more of the eight specific similar to mothers against decapentaplegic (Smad) transcription factors (Massague 1998, Mazerbourg & Hsueh 2003). The type I receptors are also designated as activin receptor-like kinases (ALKs) and are responsible, in part, for transmitting ligand specificity within target cells (Massague 1998, Lux et al. 1999). GDF-9 interacts with the BMP type II receptor (BMPRII) and the type I receptor ALK5 and activates Smad2/3 (Vitt et al. 2002, Kaivo-Oja et al. 2003, Roh et al. 2003, Mazerbourg et al. 2004), whereas BMP-2 and BMP-4 interact with BMPRII and the type I receptors, ALK3 and ALK6 which activate Smad1/5/8 (Massague 1998, Kawabata et al. 1998). However, little is know about the intracellular signaling system GDF-9 and BMP-4 in single-ovulating species such as cattle or humans. Thus, our second objective was to determine the intracellular signaling pathway used by GDF-9 and BMP-4 in bovine granulosa cells.
Materials and Methods
Biological materials
Ovaries from non-pregnant beef cows were collected from a local slaughterhouse and, based on surface diameter, follicular fluid from small (1–5 mm) and large follicles (8–22 mm) was aspirated using 20 gauge needles and 3 ml syringes and centrifuged at 200 g for 5 min to isolate granulosa cells as previously described (Langhout et al. 1991, Spicer & Chamberlain 1998, Spicer et al. 2002). This size classification was based on previous studies showing that granulosa cells from small follicles are less responsive to follicle-stimulating hormone (FSH) and IGF-I than are cells from large follicles (Spicer & Chamberlain 1998, Spicer et al. 2002), follicles 8 mm and greater have much larger estradiol concentrations than small follicles (Stewart et al. 1996, Spicer et al. 2001), and bovine follicles destined to ovulate average 10 ± 2 mm surface diameter (Marion et al. 1968). Granulosa cells were re-suspended in serum-free medium containing collagenase and DNase (Sigma Chemical Co) at 1.25 mg/ml and 0.5 mg/ml respectively, to prevent cell clumping prior to plating. Cells were maintained in this collagenase–DNase-containing medium for less than 1 h prior to dispersion in 1 ml 10% fetal calf serum (FCS) medium without proteases.
Approximately 2.0 × 105 viable cells (in 20–50 μl collagenase–DNase medium) were plated on 24-well Falcon multiwell plates (Becton Dickinson, Lincoln Park, NJ, USA) in a mixture of 1:1 Dulbecco’s modified Eagle’s medium and Ham’s F-12 containing 10% FCS, 0.12 mM gentamycin, 2.0 mM glutamine, and 38.5 mM sodium bicarbonate (all obtained from Sigma Chemical Co) as previously described (Langhout et al. 1991, Spicer & Chamberlain 1998, Spicer et al. 2002). Viability of granulosa cells from small and large follicles was determined by the trypan blue exclusion method, and averaged 58 ± 11% and 79 ± 12% respectively. Cells were cultured in an environment of 95% air and 5% CO2 at 38.5 °C in 10% FCS for the first 48 h with a medium change at 24 h. Cells were then washed twice with serum-free medium and the various treatments (see below) applied in serum-free medium for 48 h with a medium change at 24 h. After the second 24-h treatment period, medium was collected for steroid RIA and cells were collected for cell enumeration (see below).
The hormones used in the cell culture were: FSH (ovine F1913; FSH activity, 15 × NIH-FSH-S1 U/mg) from Scripps Laboratories (San Diego, CA, USA), recombinant human IGF-I and BMP-4 from R&D Systems (Minneapolis, MN, USA), and recombinant rat GDF-9 was generated and characterized as previously described (Hayashi et al. 1999, Vitt et al. 2000a). Briefly, expression vectors for wild-type and epitope-tagged GDF-9 were constructed using pcDNA3.1 Zeo (Invitrogen). N-tagged GDF-9 encoded a Flag epitope (DYKDDDDK) for the M1 antibody followed by six histidine residues fused to the amino terminus of mature GDF-9. Clonal human embryonic kidney 293T cell lines stably expressing wild-type and tagged GDF-9 were used. Quantitation of N-tagged GDF-9 was done after purification with a nickel column and measurement of protein content using Micro BCA protein assay kit (Perstorp Life Science, Rockford, IL, USA). Purified N-tagged GDF-9 was then used as a standard for the quantitation of wild-type GDF-9 in the conditioned medium (serum free) of 293T cells by immunoblots using specific GDF-9 antibodies. In experiments 1, 2, 3, and 5 this GDF-9 preparation was added at 2 μl or less per ml serum-free medium (depending on dose tested). Previous studies (Vitt et al. 2000a, Roh et al. 2003) have shown that conditioned medium from non-transfected human embryonic kidney 293T cells as well as medium with an inactive recombinant N-Tag GDF-9 has no effect on several granulosa cell functions evaluated.
Experimental design
Experiments 1 and 2 were designed to evaluate the dose–response effect of GDF-9 on proliferation and steroidogenesis of small- and large-follicle granulosa cells respectively. Cells were cultured for 48 h in 10% FCS, washed twice with serum-free medium as described earlier, and 0, 150, 300, and 600 ng/ml GDF-9 were applied for 48 h in the presence of either FSH (30 ng/ml) or FSH plus IGF-I (30 ng/ml). The doses of FSH and IGF-I were selected based on previous studies indicating that these doses are maximal for stimulation of estradiol secretion and/or cell proliferation (Spicer & Francisco 1997, Spicer & Chamberlain 1998, Spicer et al. 2002). The doses of GDF-9 were selected based on previous studies (Vitt et al. 2000a, 2002, McNatty et al. 2005a, 2005b).
Experiment 3 was designed to evaluate the effect of GDF-9 on FCS-induced cell proliferation of large-follicle granulosa cells. Cells were cultured for 48 h in 10% FCS, washed twice with serum-free medium as described earlier, and 0, 1%, and 2% of FCS was applied for 48 h in the absence or presence of GDF-9 (300 ng/ml). The dose of GDF-9 was selected based on the results of experiments 1 and 2.
Experiment 4 was designed to evaluate the effect of BMP-4 on proliferation and steroidogenesis of small- and large-follicle granulosa cells respectively. Cells were cultured for 48 h in 10% FCS, washed twice with serum-free medium as described earlier, and 0 and 30 ng/ml BMP-4 were applied for 48 h in the presence of FSH plus IGF-I (30 ng/ml). The dose of BMP-4 was selected based on previous studies indicating that this dose is maximal for its effect on steroidogenesis (Shimasaki et al. 1999, Mulsant et al. 2001, Fabre et al. 2003, Sudo et al. 2004).
Experiment 5 was designed to elucidate the specificity of the GDF-9 and BMP-4 signaling pathway and, based on findings using rat granulosa cells, we tested the ability of GDF-9 and BMP-4 to stimulate the Smad binding element (CAGA) and the BMP response element (BRE) promoter respectively in bovine granulosa cells. The CAGA and BRE promoters are known to be activated by GDF-9 mediated by Smad3 and several BMPs mediated by Smad1/5/8 respectively (Dennler et al. 1998, Kusanagi et al. 2000, Korchynskyi & ten Dijke 2002, Mazerbourg et al. 2004, Monteiro et al. 2004). Granulosa cells from small follicles were cultured as described in experiment 1 with the following treatments applied for 24 h in medium (containing 1% FCS, 30 ng/ml IGF-I, and FSH) after a 4-h transfection with the hormone-specific responsive promoters CAGA or BRE: control (no additions), GDF-9 (300 ng/ml), or BMP-4 (200 ng/ml). The doses of hormones were selected based on the results of experiment 1 and previous in vitro studies (Vitt et al. 2000a, 2002, Mazerbourg et al. 2004) indicating that these doses are effective in altering steroidogenesis and/or promoter activity in granulosa cells.
Experiment 6 was designed to evaluate the effect of purified recombinant human GDF-9 (rhGDF-9; PeproTech Inc, Rock Hill, NJ, USA) and conditioned medium (serum free) from non-transfected human embryonic kidney 293T cells on proliferation and steroidogenesis of small-follicle granulosa cells. Cells were cultured for 48 h in 10% FCS, washed twice with serum-free medium as described earlier, and 0 and 600 ng/ml rhGDF-9, 2 μl conditioned medium from non-transfected 293T cells, and/or 0 or 30 ng/ml FSH were applied for 48 h in the presence of IGF-I (30 ng/ml).
Transfection of granulosa cells
Granulosa cells (2 × 105 viable cells/well) were cultured in 24-well plates in medium supplemented with 10% FCS for 2 days as described earlier. After medium change, cells were incubated in the serum-free medium and transfected with 250 ng DNA/well using Lipofectamine 2000 (Invitrogen) as previously described (Mazerbourg et al. 2004). Briefly, the pCMV-β-galactosidase plasmid (50 ng) was co-transfected to monitor transfection efficiency. After transfection, cells were treated with GDF-9 or BMP-4 for 24 h in medium containing 1% FCS. To harvest cells, lysis buffer (200 μl; Promega Corp) was added into each well and 30 μl of the supernatant was used for luciferase determination using a luminometer (Luminark microplate reader; Bio-Rad Laboratories, Inc.). Fifty microliters of the cell lysate were also used to measure the β-galactosidase activity to monitor transfection efficiency. The reporter activity is expressed as the ratio of relative light units/β-galactosidase activity.
Determination of cell numbers and steroid concentrations
Medium was collected from individual wells and frozen at −20 °C for subsequent hormone analyses. Cells were then gently washed twice with 0.9% saline (500 μl), exposed to 500 μl trypsin solution (0.25%, w/v) for 20 min at 25 °C, and then scraped from each well. Cell aggregates were minimized by pipetting cell suspensions back and forth through a 500 μl pipette tip three to five times. Cells were then diluted in 9 ml 0.9% saline, and counted using a Coulter counter (model Zm; Coulter Electronics, Hialeah, FL, USA) as previously described (Langhout et al. 1991, Spicer & Chamberlain 1998, Spicer et al. 2002).
Concentrations of progesterone and estradiol in culture medium were determined by RIA as previously described (Langhout et al. 1991, Spicer et al. 2002). The intra- and interassay coefficients of variation were 12% and 17% for the progesterone RIA, and 7% and 14% for the estradiol RIA.
Statistical analysis
Each experiment contained three replicates per treatment and each experiment was replicated three to four times with different pools of granulosa cells. Each pool of small-follicle granulosa cells was obtained from multiple follicles collected from seven to ten cattle, and each pool of large-follicle granulosa cells was obtained from four to seven follicles collected from three to six cattle. Data are presented as the least squares means ( ± s.e.) of measurements from nine to twelve culture wells. Main effects (i.e. hormone, dose, experimental replicate) and interactions were assessed using the general linear model procedure of SAS (1999). Steroid production was expressed as ng or pg/105 cells per 24 h, and cell numbers at the termination of the experiment were used for this calculation. Specific differences in cell numbers, steroid production, and promoter (luciferase) activity among treatments were determined using Fisher’s protected least significant difference procedure (Ott 1977).
Results
Experiment 1: GDF-9 effects on small-follicle granulosa cells
Cell numbers
GDF-9 caused a dose-dependent increase (P<0.05) in basal (60% increase) and IGF-I-induced (28% increase) cell numbers (Fig. 1A).
Progesterone production
IGF-I increased (P<0.001) progesterone production (to 2-fold of controls) by granulosa cells, and GDF-9 caused a dose-dependent inhibition (P<0.05) of this increase with 600 ng/ml completely blocking the IGF-I-induced increase (Fig. 2A). At 150 ng/ml, GDF-9 had no significant effect on progesterone production (Fig. 2A). In the absence of IGF-I, only 600 ng/ml decreased (by 28%; P<0.05) progesterone production in FSH-treated cultures (Fig. 2A).
Estradiol production
IGF-I increased (P<0.01) estradiol production (to 20.7-fold of FSH-treated controls) by granulosa cells, and GDF-9 caused a dose-dependent decrease (P<0.05) in this IGF-I-induced estradiol production, with the highest dose tested (600 ng/ml) inhibiting estradiol production by 71% (Fig. 2B). At 300 ng/ml, GDF-9 inhibited (P<0.05) estradiol production by 27%. In the absence of IGF-I, none of the doses of GDF-9 affected (P>0.10) estradiol production in FSH-treated cultures (Fig. 2B).
Experiment 2: GDF-9 effects on large-follicle granulosa cells
Cell numbers
GDF-9 caused a dose-dependent increase (P<0.01) in basal (maximum 2.1-fold increase) and IGF-I-induced (maximum 2.3-fold increase) cell numbers (Fig. 1B).
Progesterone production
IGF-I increased (P<0.001) progesterone production (to 2-fold of FSH-treated controls) by granulosa cells, and GDF-9 caused a dose-dependent inhibition (P<0.05) of this increase with 600 ng/ml completely blocking the IGF-I-induced increase (Fig. 3A). In contrast to small-follicle granulosa cells, 150 ng/ml GDF-9 significantly decreased progesterone production (Fig. 3A). In the absence of IGF-I but in the presence of FSH, only 600 ng/ml decreased (by 38%; P<0.05) progesterone production (Fig. 3A).
Estradiol production
IGF-I increased (P<0.01) estradiol production (to 7.7-fold of FSH-treated controls) by granulosa cells, and GDF-9 caused a dose-dependent inhibition (P<0.05) of this increase with 300 and 600 ng/ml reducing the IGF-I-induced increase in estradiol production by 20% and 51% respectively (Fig. 3B). In the absence of IGF-I but in the presence of FSH, none of the doses of GDF-9 affected (P>0.10) estradiol production (Fig. 3B).
Experiment 3: Effect of GDF-9 on FCS-induced large-follicle granulosa cell growth
Cell numbers
GDF-9 amplified (P<0.05) basal and FCS-induced cell numbers (Fig. 4). Alone, GDF-9 increased (P<0.05) cells numbers by 2.1-fold whereas 1% and 2% FCS increased (P<0.05) cell numbers by 4.7- and 5.9-fold respectively (Fig. 4). In the presence of 1% and 2% FCS, GDF-9 further increased (P<0.05) cell numbers by 31% and 27% respectively (Fig. 4).
Experiment 4: Effects of BMP-4 on small- and large-follicle granulosa cells
Cell numbers
BMP-4 had no effect (P>0.10) on numbers of granulosa cells collected from small (control= 1.37 and BMP-4=1.54 ± 0.04 × 105 cells/well) or large (control=1.08 and BMP-4=1.22 ± 0.06 × 105 cells/well) follicles.
Experiment 5: GDF-9 and BMP-4 effects on CAGA and BRE promoter activity in small-follicle granulosa cells
Granulosa cells from small follicles were cultured for 48 h in 10% FCS, followed by transfection with either the CAGA or BRE promoter and hormonal treatments revealed that treatment with GDF-9 increased (P<0.01) CAGA promoter activity 1.8-fold of controls, whereas BMP-4 was without effect (P>0.10; Fig. 6A). In contrast, BMP-4 increased (P<0.01) BRE promoter activity by 3.9-fold of controls, whereas GDF-9 was without effect (P>0.10; Fig. 6B).
Experiment 6: Effects of rhGDF-9 and conditioned medium from 293T cells on small-follicle granulosa cells
Cell numbers
rhGDF-9 but not FSH or conditioned medium from 293T cells increased (P<0.05) the numbers of granulosa cells collected from small follicles (Table 1).
Progesterone production
FSH increased (P<0.05) progesterone production by 36% in the presence of IGF-I whereas rhGDF-9 inhibited (P<0.05) this FSH-induced increase in progesterone production (Table 1). Conditioned medium from 293T cells had no significant effect on progesterone production (Table 1).
Discussion
The results of the present study on bovine granulosa cells revealed that: (1) GDF-9 stimulates the proliferation of granulosa cells from both large and small follicles in the presence and absence of IGF-I and FCS, whereas BMP-4 was without effect; (2) GDF-9 and BMP-4 inhibit FSH-and IGF-I-induced estradiol and progesterone production by granulosa cells from both large and small follicles; (3) GDF-9 has minimal effect on granulosa cell progesterone and estradiol production induced by FSH alone; (4) GDF-9 (but not BMP-4) activates the CAGA promoter; and (5) BMP-4 (but not GDF-9) activates the BRE promoter.
In the present study, stimulatory effects of GDF-9 were observed on basal and IGF-I-induced granulosa cell proliferation whether cells were obtained from large or small follicles. These observations agree with previous reports in which treatment with GDF-9 (10–2000 ng/ml) increased numbers of rat granulosa cells (by over 2-fold) as well as cell proliferation (by 2- to 9-fold) as measured by [3H]thymidine incorporation (Vitt et al. 2000a, 2002, McNatty et al. 2005a). In contrast, a recent report indicates that treatment with either 1 or 2 μg/ml recombinant ovine GDF-9 had no significant effect on [3H]thymidine incorporation into ovine or bovine granulosa cells (McNatty et al. 2005b). In further agreement with the present study, the proliferative responses of rat granulosa cells to GDF-9 observed by Vitt et al. (2000a) was qualitatively similar whether cells were obtained from small antral follicles or large preovulatory follicles, although the maximally effective dose of GDF-9 was 5-fold lower in rat granulosa cells obtained from large preovulatory follicles. The latter suggests that rat granulosa cells of mature differentiated follicles may be more sensitive to GDF-9 than those of immature undifferentiated follicles. Data in the present study support this assumption because large-follicle granulosa cell proliferation and progesterone production (see next paragraph) were significantly affected by the lowest dose of GDF-9 tested, whereas small-follicle granulosa cells were not. Interestingly, BMP-4 at a dose effective in inhibiting steroidogenesis had no effect on IGF-I-induced cell numbers in the present study. Shepherd & Nachtigal (2003) reported that neither 2- nor 4-day BMP-4 treatment affected FCS-induced proliferation of four types of human ovarian carcinoma cells. Also, others have reported that BMP-4 (Fabre et al. 2003) and BMP-2 (Souza et al. 2002) have no effect on ovine granulosa cell proliferation while significantly affecting steroidogenesis in vitro. Thus, the present study supports the notion that two TGF-β superfamily members that bind to the same BMPRII receptor (Kawabata et al. 1998, Lux et al. 1999, Newfeld et al. 1999) but derived from two different (i.e. oocyte vs theca) cell types have diverse biological responses within the granulosa layer, the latter of which are mediated by the various type I receptors (ALKs) and intracellular Smad response proteins (Massague 1998, Mazerbourg & Hsueh 2003, Mazerbourg et al. 2004). Whether GDF-9 and BMP-4 have stimulatory effects on preantral follicular growth in cattle as they do in rodents (Vitt et al. 2000b, Nilsson & Skinner 2002, 2003, Latham et al. 2004, Wang & Roy 2004) and humans (Hreinsson et al. 2002) awaits further elucidation. Also, whether GDF-9 plays a role in the regulation of ovulation rate in cattle, as recently demonstrated for sheep (Hanrahan et al. 2004, Juengel et al. 2004), will require additional research.
Previous studies with rat granulosa cells have reported that 48-h (Vitt et al. 2000a, 2002) and 6-day (McNatty et al. 2005a) treatment with>30 ng/ml GDF-9 inhibits progesterone production induced by FSH. Similarly, in the present study, GDF-9 decreased bovine granulosa cell progesterone production in the presence of FSH alone (highest dose of GDF-9 only) or a combination of FSH and IGF-I (all doses of GDF-9). McNatty et al. (2005b) recently reported that 6-day treatment with 2 μg/ml GDF-9 inhibited progesterone production by ovine and bovine granulosa cells cultured in the presence of FSH, IGF-I, and insulin. Yamamoto et al.(2002) also observed that GDF-9 inhibits progesterone production induced by 8-bromo-cAMP in cultured human granulosa cells. In the present study, under basal conditions (the presence of FSH), GDF-9 (only 600 ng/ml) had a weak inhibitory effect on progesterone production. In previous studies with cultured mouse granulosa cells, 50–100 ng/ml GDF-9 treatment for 16–24 h increased basal progesterone production but had no effect on FSH-induced progesterone production (Elvin et al. 1999, 2000). Discrepancies among studies could be due to species differences and/or differences in culture conditions (e.g. duration of treatment or presence or absence of FCS). Consistent with previous reports (Spicer & Chamberlain 1998, Spicer et al. 2002), IGF-I stimulated progesterone and estradiol production by bovine granulosa cells in the presence of FSH (Figs 2 and 3). It should be emphasized that granulosa cells in the present and previous studies, because of serum exposure (Orly et al. 1980, Luck et al. 1990), may have partially luteinized and therefore be exhibiting some luteal cell activity. The less than 5-fold increase in estradiol secretion induced by a high dose (i.e. 30 ng/ml) of FSH combined with the very high ratio (i.e.>150) of progesterone:estradiol secreted by the granulosa cells of the present study supports this latter suggestion.
Reported for the first time using bovine granulosa cells, GDF-9 inhibited estradiol secretion by granulosa cells obtained from large and small follicles of a single-ovulating species (Figs 2B and 3B). Previously, treatment of rat granulosa cells with GDF-9 (30–150 ng/ml) inhibited (by up to 58%) FSH-induced estradiol production without affecting forskolin-induced estradiol production (Vitt et al. 2000a). Thus, granulosa cell aromatase activity from multiple- and single-ovulating species appear to respond similarly to GDF-9. Also consistent with the present study, Vitt et al. (2000a) found that the inhibitory effect of GDF-9 on estradiol production was similar whether cells were obtained from small antral or large preovulatory rat follicles. Collectively, these studies indicate that GDF-9 is an effective suppressor of aromatase activity regardless of the size (or differentiation state) of the follicle in which the granulosa cells reside.
Similar to GDF-9, BMP-4 inhibited both progesterone and estradiol production by bovine granulosa cells from small and large follicles. Although originally identified for their ability to induce bone and cartilage formation (Wan & Cao 2005), BMPs have since been shown to play important roles in cellular differentiation and formation in many other tissues and organs including the ovary (Massague 1998, Knight & Glister 2003, Mazerbourg & Hsueh 2003). Inhibitory effects of BMP-4 on progesterone production by granulosa cells of sheep (Mulsant et al. 2001, Fabre et al. 2003, Pierre et al. 2004), cattle (Glister et al. 2004), and rats (Shimasaki et al. 1999, Sudo et al. 2004) have been documented. The present study further evaluated BMP-4 effects on aromatase activity. In cultured rat granulosa cells, BMP-4 stimulates FSH-induced estradiol production (Shimasaki et al. 1999, Sudo et al. 2004) but its effect in the presence of IGF-I was not studied. One other report indicated that a 6-day treatment of bovine granulosa cells with BMP-4 increases estradiol production and cell proliferation (Glister et al. 2004) but because estradiol production was not corrected for increased cell numbers, clear interpretation of these data is not possible. In the present study, BMP-4 suppressed FSH plus IGF-I-induced aromatase activity as well as progesterone production in bovine granulosa cells regardless of whether cells originated from small or large follicles. More extensive studies are needed to determine if responsiveness to BMP-4 changes during follicular development and if locally produced BMPs change during follicular development in cattle.
Using bovine granulosa cells, we have shown that GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter. Similar results has been obtained in rat and human granulosa cells (Kaivo-Oja et al. 2003, 2005, Roh et al. 2003, Mazerbourg et al. 2004, Sudo et al. 2004). Previous studies in rat and human granulosa cells have suggested that GDF-9 induces the phosphorylation of Smad2/3 after interaction with the receptor type II, BMPRII, and the receptor type I, ALK5 (Vitt et al. 2002, Mazerbourg et al. 2004, Kaivo-Oja et al. 2005). Stimulation of the CAGA promoter is mediated by TGF-β, activin, or GDF-9 after phosphorylation of the downstream Smad3 proteins, whereas BMP-2 and BMP-7 activate Smad1, 5, and 8 (Massague 1998) and subsequently the BRE and GCCG promoters (Kusanagi et al. 2000, Korchynskyi & ten Dijke 2002). However, the distinct biological effects of the TGF-β superfamily members may be exerted by the transcriptional regulation of target genes in a cell-type specific manner that may involve ALK-specific and opposing effects that may include the inhibitory Smads (Smad6 and Smad7) (ten Dijke & Hill 2004) and may explain how factors such as GDF-9 have both stimulatory and inhibitory effects on granulosa cell functions. Further studies will be required to elucidate the elements involved in the inhibitory (steroidogenesis) versus stimulatory (cell proliferation) action of GDF-9.
Effects of recombinant human GDF-9 (rhGDF-9) and conditioned medium (CM: from non-transfected human embryonic kidney 293T cells) on granulosa cell numbers, and on progesterone and estradiol production
| No. of samples | Cell numbers (× 105/well) | Progesterone (ng/105 cells/24 h) | Estradiol (pg/105 cells/24 h) | |
|---|---|---|---|---|
| Granulosa cells from small (1 to 5 mm) bovine follicles were cultured as described in Materials and Methods, and treated for 48 h with FSH (30 ng/mL), CM (2 μl/well), and/or 600 ng/mL of rhGDF-9 . | ||||
| *All treatments were applied concomitantly with 30 ng/mL of IGF-I. abcWithin a column, means without a common letter differ (P<0.05). | ||||
| Treatment* | ||||
| Control | 9 | 0.61a | 42.0a | 46a |
| +FSH | 9 | 0.77b | 57.1b | 216c |
| +FSH +CM | 9 | 0.84b | 56.2b | 248c |
| +FSH +rhGDF-9 | 9 | 1.06c | 37.9a | 130b |
| s.e.m. | 0.05 | 4.3 | 17 | |
Stimulatory effect of 2-day treatment of various doses of GDF-9 in the presence of FSH (30 ng/ml) with or without IGF-I (30 ng/ml) on numbers of granulosa cells from small (1–5 mm) (A) and large (8–22 mm) (B) bovine follicles. Within a panel, means without a common letter differ (P<0.05; n = 3 experiments).
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06503
Inhibitory effect of 2-day treatment of various doses of GDF-9 in the presence of FSH (30 ng/ml) with or without IGF-I (30 ng/ml) on progesterone (A) and estradiol (B) production by granulosa cells from small (1–5 mm) bovine follicles. Within a panel, means without a common letter differ (P<0.05; n = 3 experiments).
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06503
Inhibitory effect of 2-day treatment of various doses of GDF-9 in the presence of FSH (30 ng/ml) with or without IGF-I (30 ng/ml) on progesterone (A) and estradiol (B) production by granulosa cells from large (8–22 mm) bovine follicles. Within a panel, means without a common letter differ (P<0.05; n = 3 experiments).
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06503
Stimulatory effect of 2-day treatment of GDF-9 (300 ng/ml) on FCS-induced granulosa cell proliferation. Granulosa cells from large (8–22 mm) bovine follicles were cultured for 2 days in the presence of 0, 1, or 2% FCS. Means without a common letter differ (P<0.05; n = 3 experiments).
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06503
Inhibitory effect of 2-day treatment of BMP-4 (30 ng/ml) in the presence of FSH (30 ng/ml) and IGF-I (30 ng/ml) on progesterone (A) and estradiol (B) production by granulosa cells from large (8–22 mm) and small (1–5 mm) bovine follicles. Means without a common letter differ (P<0.05; n = 4 experiments).
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06503
Stimulatory effect of 24-h treatment of GDF-9 (but not BMP-4) (A) on CAGA promoter activity and BMP-4 (but not GDF-9) (B) on BRE promoter activity in granulosa cells from small (1–5 mm) bovine follicles. Means without a common letter differ (P<0.05). β-Galac, β-galactosidase.
Citation: Journal of Endocrinology 189, 2; 10.1677/joe.1.06503
The authors thank M Aleman and C Klein for technical assistance, Creekstone Farms (Arkansas City, KS, USA) for their generous donations of bovine ovaries, Dr C H Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden) for the CAGA promoter–luciferase construct, Dr O Korchynskyi (University of North Carolina, Chapel Hill, NC, USA) for the BRE promoter–luciferase construct, and Dr R Matts (Oklahoma State University, Stillwater, OK, USA) for use of the luminometer.
Funding This research was supported in part under project H-2510 of the Oklahoma Agricultural Experiment Station, and by the National Research Initiative Competitive Grant no. 2005–35203–15334 from the USDA Cooperative State Research, Education, and Extension Service. This paper was approved for publication by the Director, Oklahoma Agricultural Experimental Station. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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