REPRODUCTIONRESEARCH

Differential actions of fibroblast growth factors on intracellular pathways and target gene expression in bovine ovarian granulosa cells

Zhongliang Jiang1,2 and Christopher A Price2 1College of Animal Science and Technology, Northwestern A&F University, Yangling, ShaanXi, China and 2Faculte´ de Me´decine Ve´te´rinaire, Centre de Recherche en Reproduction Animale, Universite´ de Montre´al, CP 5000, St-Hyacinthe, Quebec, Canada J2S 7C6 Correspondence should be addressed to C A Price; Email: [email protected]

Abstract

Several fibroblast growth factors (FGFs), including FGF1, FGF4 and FGF10, alter ovarian granulosa cell function. These ligands exhibit different patterns of receptor activation, and their mechanisms of action on granulosa cells remain unknown. The objective of this study was to identify the major pathways and target genes activated by FGF1, FGF4 and FGF10 in primary oestrogenic granulosa cells cultured under serum-free conditions. FGF1 and FGF4 increased levels of mRNA encoding Sprouty family members, SPRY2 and SPRY4, and the orphan nuclear receptors NR4A1 and NR4A3. Both FGF1 and FGF4 decreased levels of mRNA encoding SPRY3 and the pro-apoptotic factor BAX. FGF1 but not FGF4 stimulated expression of the cell cycle regulator, GADD45B. In contrast, FGF10 altered the expression of none of these genes. Western blot demonstrated that FGF4 activated ERK1/2 and Akt signalling rapidly and transiently, whereas FGF10 elicited a modest and delayed activation of ERK1/2. These data show that FGF1 and FGF4 activate typical FGF signalling pathways in granulosa cells, whereas FGF10 activates atypical pathways. Reproduction (2012) 144 625–632

Introduction whereas other pathways and actions of FGF vary between cell types (Dailey et al. 2005). The predominant Fibroblast growth factors (FGFs) have been shown to play pathways of FGF action in ovarian cells have received various roles in the regulation of reproductive processes. little attention; FGF2 activated PKC in rat granulosa FGFs belong to a family of 22 closely related proteins cells (Peluso et al. 2001) and increased ERK1/2 and (Itoh & Ornitz 2004), most of which signal through Akt phosphorylation in bovine granulosa cells (Jiang transmembrane receptor tyrosine kinases. In the ovary, et al. 2011). the FGF that has been the attention of most research is Activation of these pathways leads to expression of FGF2. The FGF2 gene is predominantly expressed in the FGF target genes, including the Sprouty (SPRY) family of theca cell layer (Koos & Olson 1989, Stirling et al. 1991, negative regulators of FGFR tyrosine kinase activity Berisha et al. 2000) and acts upon granulosa cells. FGF2 (Cabrita & Christofori 2008). In the ovary, FGF2 induced has been demonstrated to promote granulosa cell SPRY2 expression in human granulosa–luteal cells and proliferation, prevent apoptosis and decrease steroido- mouse cumulus cells (Haimov-Kochman et al. 2005, genesis in a number of species (Gospodarowicz & Sugiura et al. 2009) and increased levels of mRNA Bialecki 1979, Baird & Hsueh 1986, Yamoto et al. encoding both SPRY2 and SPRY4 in bovine granulosa 1993, Lavranos et al. 1994, Vernon & Spicer 1994, Cao cells (Jiang et al. 2011). et al. 2006). FGF2 is not the only FGF shown to act on granulosa The mechanism of action of FGFs in the ovary has not cells. FGF1 is similar to FGF2 in that it is also expressed been extensively explored. Studies from a variety of non- predominantly in theca cells in cattle (Berisha et al. reproductive tissues and cell-lines have demonstrated 2004) and is mitogenic in granulosa and theca cells that FGF receptor (FGFR) activation induces intracellular (Roberts & Ellis 1999). FGF1 has been shown to induce signalling through MAPKs, protein kinase C (PKC) and transient expression of SPRY2 in osteoblast cells (Yang phosphatidylinositol-3-kinase (PI3K) and Akt activation et al. 2006), although its target genes in granulosa cells (Dailey et al. 2005, Iwata & Hevner 2009). FGF are unknown. Two other FGFs have been shown to alter signalling through MAPK appears to be ubiquitous SPRY expression in a reproductive tissue; FGF4 and

q 2012 Society for Reproduction and Fertility DOI: 10.1530/REP-12-0199 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/30/2021 10:31:07PM via free access 626 Z Jiang and C A Price

FGF10 increased SPRY2 mRNA abundance in human (Fig. 1). The addition of 10 ng/ml FGF4 increased placenta (Anteby et al. 2005), but curiously neither FGF4 abundance of mRNA encoding SPRY2 and SPRY4 but nor FGF10 increased SPRY2 mRNA levels in human had no effect on mRNA encoding SPRY1 or SPRY3 luteal cells (Haimov-Kochman et al. 2005). Theca cells (Fig. 1). For both FGFs, the effect on SPRY2 mRNA levels express FGF10 in cattle, and FGF10 inhibited oestradiol was rapid, with a significant increase within 1 h, (E2) secretion from granulosa cells in vitro and in vivo whereas the effects on SPRY4 were slower, not reaching (Buratini et al. 2007, Gasperin et al. 2012). significance until 4 h. In contrast, FGF10 had no effect A comparison of FGF1, FGF4 and FGF10 could offer on SPRY mRNA levels (Fig. 1). Addition of FSH also had insight into differential FGF signalling, as they have no effect on SPRY mRNA levels (data not shown). different receptor (FGFR) activation patterns. There are The effects of FGF1 and FGF4 on SPRY expression seven FGFR proteins derived from four genes through were dose dependent. When challenged for 2 h, both alternative splicing events; each splice variant has FGFs increased levels of mRNA for SPRY1, SPRY2 and specific ligand binding affinities (Zhang et al. 2006). SRPY4 and decreased those encoding SPRY3 (Fig. 2). Bovine granulosa cells express all receptors except Even at the highest dose tested, FGF10 did not alter SPRY FGFR4 (Buratini et al. 2005, Berisha et al. 2006). FGF1 mRNA abundance. activates all six granulosa receptors, FGF4 activates the We then determined the effects of these FGFs on other ‘c’ splice variants (FGFR1c, FGFR2c and FGFR3c), and FGF target genes, specifically members of the NR4A FGF10 activates ‘b’ splice variants, predominantly orphan nuclear receptors and the ETS transcription factor FGFR2b (Zhang et al. 2006). The objective of this study family. Addition of graded doses of FGF1 and FGF4 for was to determine whether FGF1, FGF4 and FGF10 have 2 h caused significant increases in abundance of mRNA divergent signalling pathways and target genes in non- encoding NR4A1 and NR4A3 but had no effect on luteinizing bovine granulosa cells. The target genes NR4A2 mRNA levels (Fig. 3). Even at the highest dose studied were early response genes of the SPRY, ETV and tested, FGF10 did not stimulate NR4A mRNA NR4A families known to be regulated in granulosa cells abundance. (Jiang et al. 2011) as well as other cell types and growth FGF1 at 10 ng/ml caused a transient increase in ETV5 and survival genes known to be regulated in various mRNA levels (Fig. 4) but did not alter abundance of cancer cells (Karsan et al. 1997, Cosgrave et al. 2006). mRNA encoding ETV1 or ETV4 (PO0.1; not shown). The addition of FGF4 did not affect abundance of mRNA Results encoding ETV1 or ETV4 (not shown) and almost doubled the abundance of ETV5 mRNA, but this did not reach FGF1 and FGF4 regulate gene expression in granulosa significance (Fig. 4). FGF10 had no effect on abundance cells of mRNA encoding ETV1, ETV4 or ETV5. The addition of 10 ng/ml FGF1 increased the abundance The effects of FGFs on apoptosis and cell cycle genes of mRNA encoding SPRY2 and SPRY4 (P!0.01), were also measured. FGF1 stimulated abundance of decreased the abundance of mRNA encoding SPRY3 mRNA encoding GADD45B and decreased BAX mRNA (P!0.05) and had no effect on SPRY1 mRNA levels levels (Fig. 4), while having no effect on BIRC5 or BCL2

SPRY1 SPRY2 4 4 b b abab b 3 3 ab c c 2 2 a t) C a 1 1

01248 01248 01248 01248 01248 01248

c 4 SPRY34 SPRY4 bc

ab 3 FGF1 3 b ab FGF4 ab a Relative mRNA abundance (dd mRNA abundance Relative Figure 1 Effect of FGF1, FGF4 and FGF10 on SPRY 2 FGF10 2 ab a ab ab mRNA abundance in bovine granulosa cells in ab ab a serum-free culture. Cells were challenged on day 5 1 b 1 of culture with 10 ng/ml FGF for the times given. Data are meansGS.E.M. of three independent 01248 01248 01248 01248 01248 01248 replicates. For each treatment, means with common Time (h) Time (h) or no letters are not significantly different (P!0.05).

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7 SPRY17 SPRY2 c 6 6 c c c 5 5

t) 4 4 b C 3 b 3 b ab b b b 2 a 2 a a a a 1 1

0 1 10 50 0 1 10 50 0 11050 0 11050 0 11050 0 11050 7 SPRY37 SPRY4 6 6 FGF1 5 FGF4 5 4 FGF10 4 b Figure 2 Effect of dose of FGF1, FGF4 and FGF10 Relative mRNA abundance (dd 3 3 b b ab ab on SPRY mRNA abundance in bovine granulosa 2 2 a ab a cells. Cells were challenged on day 5 of culture for a ab ab ab ab a 1 b b 1 2 h with the doses of FGF given. Data are mean GS.E.M. of three independent replicates. For each 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 0 1 10 50 treatment, means with common or no letters are Fibroblast growth factor (ng/ml) not significantly different (P!0.05). mRNA levels (not shown). FGF4 decreased BAX mRNA SPRY1, SPRY2 and SPRY4 mRNA abundance. FGF4 levels without effect on GADD45B (Fig. 4), BIRC5 or resulted in a significant and transient increase in Akt BCL2 mRNA levels (not shown), and FGF10 did not alter phosphorylation (Fig. 6C), although this was not as abundance of mRNA of any of these genes. robust or as prolonged as that observed for pERK1/2. In To verify that the FGF10 was biologically active in our cultures with FGF10, pAkt was not consistently detected cell model, we cultured cells for 6 days and added irrespective of time after treatment (not shown). 10 ng/ml FGF1 or FGF10 for the last 4 days of culture. FGF1 and FGF10 significantly reduced E2 concentrations compared with control (2.5G0.3, 2.0G0.3 and 8.0 Discussion G1.9 ng/ml respectively; P!0.05). FGFs act on ovarian granulosa cells to inhibit steroid secretion, but the mechanisms involved are not clear. Intracellular pathways activated by FGF in FGF2 activates the typical FGF pathway in granulosa granulosa cells cells, through pERK1/2 (Jiang et al. 2011), as do FGF1 and FGF4 (this study). However, the major finding of this We further explored the discrepancy between the effects study is that FGF10 signalling differs from that of the of FGF4 and FGF10. To verify whether both activate the other FGFs and appears not to employ typical FGF MAPK pathway in granulosa cells, abundance of signalling pathways. intracellular phosphorylated ERK1/2 was measured by Activation of ERK1/2 and increased expression of western blot. FGF4 significantly increased the levels of SPRY genes are generally considered to be typical pERK1/2 within 5 min (Fig. 5). In contrast, there was a responses to FGFs (Bottcher & Niehrs 2005, Dailey weak gradual increase in pERK1/2 protein after et al. 2005, Iwata & Hevner 2009), as observed for FGF2 challenge with FGF10 (Fig. 5); there was no main effect in bovine granulosa cells (Jiang et al. 2011). In this study, of time by ANOVA, but post hoc comparison of 120 and FGF10 decreased E2 secretion from granulosa cells, as 240 min time points with the time 0 control (orthogonal previously observed (Buratini et al. 2007, Gasperin et al. contrasts) indicated a significant difference (P!0.05; 2012), but did not result in rapid phosphorylation of Fig. 5). ERK1/2 or acute upregulation of any SPRY gene studied. As FGF4 increased ERK1/2 phosphorylation, we This is in contrast to the activation of ERK1/2 by FGF10 explored further intracellular signalling by FGF4. observed in endometrial carcinoma cells, human and rat Inhibition of PI3K significantly inhibited basal SPRY1, alveolar epithelial cells and in the developing mouse SPRY2 and SPRY3 mRNA levels (Fig. 6). FGF4 increased prostate bud (Taniguchi et al. 2003, Upadhyay et al. SPRY1, SPRY2 and SPRY4 mRNA abundance, and 2005, Kuslak & Marker 2007), and the FGF10-induced cotreatment with PI3K inhibitor blocked the effect of increase in SPRY2 mRNA levels in the mouse lung FGF4 (Fig. 6A). As before, FGF4 did not stimulate SPRY3 epithelium and the human placenta (Tefft et al. 2002, mRNA levels. Pretreatment of cells with the PKC Anteby et al. 2005, Natanson-Yaron et al. 2007). Thus, inhibitor GF109203X reduced basal SPRY expression, granulosa cells appear to be unusual in that the although this reached significance for SPRY1 only inhibitory effect of FGF10 on E2 secretion does not (Fig. 6B). Inhibition of PKC reduced FGF4-stimulated involve acute stimulation of ERK1/2 or the typical FGF www.reproduction-online.org Reproduction (2012) 144 625–632

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b cells (Haimov-Kochman et al. 2005), and this may be 5 NR4A1 explained by different patterns of FGFR expression in b these cells; in cattle at least, granulosa cells express 4 ab b FGFR3c whereas luteal cells do not (Guerra et al. 2008). ab Of some interest is the inhibitory effect of FGF1 and, to a 3 a lesser extent, FGF4 on SPRY3 mRNA levels, which was not previously observed for FGF2 (Jiang et al. 2011). To 2 a a our knowledge, the one other report describing 1 regulation of SPRY3 expression comes from a study of Xenopus embryos involving brain-derived neurotropin factor and not FGF signalling (Panagiotaki et al. 2010). 0 1 10 50 0 1 10 50 0 1 10 50 Other putative FGF response genes are not well

t) 5 described in mammals. In lower vertebrates, FGF1 and C FGF1 NR4A2 4 FGF4 3 ETV5 FGF10 3 2 2 b b

ab ab 1 1 a

Relative mRNA abundance (dd 0 1 10 50 0 1 10 50 0 1 10 50

01248 01248 01248 5

b t) b b NR4A3 GADD45B 4 C 2 FGF1 b ab b ab FGF4 3 ab FGF10 a 2 a a a a 1 1

0 1 10 50 0 1 10 50 0 1 10 50

Fibroblast growth factor (ng/ml) Relative mRNA abundance (dd 01248 01248 01248 Figure 3 Effect of FGF1, FGF4 and FGF10 on abundance of mRNA encoding the orphan nuclear receptors NR4A1, NR4A2 and NR4A3 2 in bovine granulosa cells in serum-free culture. Cells were challenged BAX on day 5 of culture for 2 h with the doses of FGF given. Data are meanGS.E.M. of three independent replicates. For each treatment, means with common or no letters are not significantly different (P!0.05). a 1 a a a ab ab early-response genes, or that there is a species-specific ab b ab difference in FGF10 signalling. b Addition of FGF1 and FGF4 to cultured granulosa cells resulted in rapid and dose-dependent increases in mRNA encoding SPRY2 and SPRY4 in a manner similar to that 01248 01248 01248 previously observed for FGF2 (Jiang et al. 2011). Higher Time (h) doses of FGF1 were required to increase SPRY1 mRNA levels compared with those needed to stimulate SPRY2 Figure 4 Effect of FGF1, FGF4 and FGF10 on abundance of mRNA or SPRY4, which is also compatible with the less robust encoding the transcription factor ETV5, the cell cycle regulator regulation of SPRY1 observed for FGF2 (Jiang et al. GADD45B and the apoptosis-associated protein BAX. Bovine granulosa cells were cultured in serum-free medium for 5 days and 2011). The sixfold stimulation of SPRY2 mRNA by 50 ng/ then challenged with 10 ng/ml FGF for the times given. Data are ml FGF4 observed here is in contrast with the lack of meanGS.E.M. of three independent replicates. For each treatment, effect of the same dose of FGF4 reported in human luteal means with common or no letters are not significantly different (P!0.05).

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FGF4 FGF10 FGF on survival/death genes appear to be cell type and ligand specific. ERK1/2 As FGF10 did not stimulate any of the target genes pERK1/2 examined, we could not use inhibitors to explore the b intracellular pathways activated by this growth factor. However, experiments with FGF4 indicated that inhibi- b 3 tion of PI3K or PKC pathways completely inhibited the * ability of FGF4 to stimulate SPRY1 and SPRY4 mRNA * levels and attenuated the stimulation of SPRY2. These b b 2 b results are in agreement with the effects of FGF2 in b granulosa cells (Jiang et al. 2011), consistent with the similar receptor activation pattern of these two ligands 1 a pERK1/2 : ERK1/2 ratio A SPRY1SPRY2 SPRY3 SPRY4

6 c 0 5 15 30 60 120 240 0 5 15 30 60 120 240 5 Time (min) 4 c 3 d b Figure 5 FGF4 and FGF10 regulation of ERK1/2 phosphorylation. abundance a a a Relative mRNA 2 Bovine granulosa cells were cultured in serum-free medium for 5 days a a a and then challenged with 10 ng/ml FGF4 or FGF10 for the times given. 1 b b b b b a One representative western blot for phospho- and total ERK1/2 is shown. Data are meanGS.E.M. of three independent replicates. Means CLFFLCLFFLCLFFLCLFFL without common letters are significantly different (P!0.05); asterisks denote difference from time 0 by orthogonal contrasts. B SPRY1SPRY2 SPRY3 SPRY4 b FGF4 were shown to stimulate expression of ETV4 6 (McCabe et al. 2006, Eloy-Trinquet et al. 2009), but this 5 c was not observed in this study. FGF2 stimulated ETV5 4 c b mRNA levels in bovine granulosa cells (Jiang et al. 3 2011), and this study shows that FGF1 had a similar 2 a ab abundance ab a a effect. FGF4 stimulated ETV5 expression in the chick Relative mRNA 1 b a a embryo (Firnberg & Neubuser 2002), and a trend in this direction was observed in granulosa cells, but this did CGFFGCGFFGCGFFGCGFFG not reach significance. In this study, FGF1 and FGF4 Treatment group stimulated levels of mRNA encoding the orphan nuclear receptors NR4A1 and NR4A3, in partial agreement with C Akt the effect of FGF2 on NR4A1 expression in bovine pAkt granulosa cells (Jiang et al. 2011). We are not aware of 4 * other reports describing regulation of NR4A family * members by FGF1 or FGF4. 3 Some members of the FGF family are mitogenic and/or 2 anti-apoptotic, including FGF1 (Roberts & Ellis 1999); therefore, we assessed the effects of FGF1 and FGF4 on 1 pAkt : Akt ratio select death/survival genes. FGF1 and FGF4 decreased abundance of mRNA encoding the pro-apoptotic protein 0 5 15 30 60 120 240 BAX, whereas we previously showed that FGF2 did not Time (min) alter BAX mRNA (Jiang et al. 2011), and FGF1 increased Figure 6 Involvement of the PI3K and PKC signalling pathways in FGF4 levels of mRNA encoding the cell cycle regulator stimulation of SPRY mRNA abundance. Cells were challenged for 2 h GADD45B, consistent with a similar effect of FGF2 with FGF4 (10 ng/ml) with and without pretreatment with PI3K in granulosa cells (Jiang et al. 2011). Neither FGF1, inhibitor LY294002 (panel A) or PKC inhibitor GF109203X (panel B). FGF4 (this study) nor FGF2 (Jiang et al.2011) Phosphorylation of Akt in response to FGF4 was measured by western G altered abundance of mRNA encoding the cell survival blot (panel C). Data are mean S.E.M. of three independent replicates. For each mRNA, means with common or no letters are not significantly genes BIRC5 and BCL2 in granulosa cells, which is different (P!0.05); asterisks in panel C denote difference from time 0. in contrast to effects of FGF in cancer cells (Karsan C, control; L, LY294002; F, FGF4; G, GF109203X; FL, FGF4C et al. 1997, Cosgrave et al. 2006). Thus, the effects of LY294002; FG, FGF4CGF109203X. www.reproduction-online.org Reproduction (2012) 144 625–632

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(Zhang et al. 2006). These inhibitors also attenuated SPRY2 and SPRY4 mRNA. As a negative control for SPRY SPRY mRNA abundance in the absence of FGF, which expression, cells were also challenged with 10 ng/ml FSH for we have previously observed (Jiang et al. 2011), and the same periods examined for FGFs. Specific signalling which suggests that PI3K and PKC pathways are active inhibitors were also used to dissect the major signalling in these cells, although we cannot rule out potential pathway activated by FGF4. The inhibitors were GF109203X, non-specific effects of these inhibitors on these cells. an inhibitor of PKC (3 mmol; Sigma–Aldrich), and LY294002, a Taken together, these data demonstrate that FGF1, PI3K inhibitor (20 mmol). Inhibitors were dissolved in dimethyl which activates all FGFRs, and FGF4, which activates sulfoxide (DMSO) and added directly to the medium. The cells the ‘c’ splice variants, had similar actions on ovarian were pretreated with inhibitors for 1 h before adding FGF4 for a granulosa cells and activated typical FGF target genes. In further 2 h. Controls were treated with DMSO. Each pool of contrast, FGF10, which activates only FGFR2b, did not cells collected on a specific day constituted one replicate, and all experiments were performed with three independent pools activate the typical FGF target genes. The mechanism of of cells. For logistical reasons, treatments (FGF1, FGF4 or action of FGF10 remains to be determined. FGF10) were not applied to the same replicate. To verify that FGF10 was biologically active, 10 ng/ml Materials and Methods FGF10 was included in the medium added on days 2 and 4, and E2 concentrations in medium on day 6 were compared Cell culture with cells treated with FGF1 (10 ng/ml) or with medium alone. All materials were obtained from Invitrogen Life Technologies unless otherwise stated. Bovine granulosa cells were cultured Total RNA extraction and real-time RT-PCR in serum-free conditions under which E2 and progesterone secretion increase with time over 6 days and are responsive to After treatments, the culture medium was removed and total FSH (Gutie´rrez et al. 1997). FSH stimulates abundance of RNA was extracted using TRIzol according to the manufac- oestrogenic enzymes (Silva & Price 2000, Sahmi et al. 2004) turer’s instructions. Total RNA was quantified by absorbance through a predominantly PKA/cAMP pathway (Silva et al. at 260 nm. RT was performed on 1 mg DNase-treated total 2006), and there is little evidence of luteinization as RNA in the presence of 1 mmol/l oligo(dT) primer and 4 U determined by the lack of increase in mRNA encoding STAR Omniscript RTase (Qiagen), 0.25 mmol/l dideoxynucleotide protein (Sahmi et al. 2004, Zheng et al. 2008). These cells triphosphate (dNTP) mix and 19.33 U RNase Inhibitor (GE respond to FGF2 and FGF10 with dose-dependent decreases in Healthcare, Baie D’Urfe´, QC, Canada) in a volume of 20 ml steroid secretion when added for 6 days of culture (Cao et al. at 37 8C for 1 h. The reaction was terminated by incubation 2006, Buratini et al. 2007). at 93 8C for 5 min. Bovine ovaries were obtained from adult cows, irrespective Real-time PCR was performed on a 7300 Real-Time PCR of stage of the oestrous cycle, at an abattoir and transported to system (Applied Biosystems, Streetsville, ON, Canada) with the laboratory at 30 8C in PBS containing penicillin Power SYBR Green PCR Master Mix. The bovine-specific (100 IU/ml), streptomycin (100 mg/ml) and fungizone primers for target genes have previously been published (1 mg/ml). Granulosa cells were harvested from follicles (Jiang et al. 2011). Common thermal cycling parameters 2–5 mm diameter, and the cell suspension was filtered through (3 min at 95 8C, 40 cycles of 15 s at 95 8C, 30 s at 59 8C and a 150 mesh steel sieve (Sigma–Aldrich Canada). Cell viability 30 s at 72 8C) were used to amplify each transcript. Melting was assessed by Trypan dye exclusion. Cells were seeded into curve analyses were performed to verify product identity. 24-well tissue culture plates (Sarstedt, Inc., Newton, NC, USA) Samples were run in duplicate and were expressed relative at a density of 106 viable cells in 1 ml DMEM/F12 containing to the histone H2AFZ as housekeeping gene. This gene is sodium bicarbonate (10 mmol/l), sodium selenite (4 ng/ml), routinely used in our laboratory and shows similar stability BSA (0.1%; Sigma–Aldrich), penicillin (100 U/ml), strepto- to cyclophilin A, both of which were more stable in mycin (100 mg/ml), transferrin (2.5 mg/ml), nonessential amino granulosa cells than glyceraldehyde-3-phosphate dehydro- acid mix (1.1 mmol/l), bovine insulin (10 ng/ml), androstene- genase as determined by geNorm Software (Ghent, Belgium) K K dione (10 7 M at start of culture and 10 6 M at each medium (Ramakers et al. 2003). Data were normalized to a calibrator change) and bovine FSH (10 ng/ml; AFP5346D; National sample using the DDCt method with correction for Hormone and Peptide Program, Torrance, CA, USA). Cultures amplification efficiency (Pfaffl 2001). were maintained at 37 8Cin5%CO2, 95% air for 6 days, with 70% medium being replaced on days 2 and 4. Western blotting After challenge with FGF4 or FGF10, cells were washed with Experimental treatments cold PBS and lysed in 100 ml/well cold RIPA buffer (25 mM To assess the early responses of granulosa cells to FGFs, cells Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium were treated on day 5 of culture with graded doses of human deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate and recombinant FGF1, FGF4 or FGF10 (PeproTech, Rocky Hill, NJ, protease inhibitor cocktail). The lysate was centrifuged at USA) for 2 h, or with 10 ng/ml FGF for different periods. The 6000 g for 5 min at 4 8C. The resulting supernatant was retained 2-h time point was chosen based on the time-course and stored at K20 8C. Protein concentrations were determined experiment as appropriate for detecting increases in both by BCA protein assay (Pierce; Rockford IL, USA).

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Samples were resolved on 10% SDS–polyacrylamide gels Acknowledgements (15 mg total protein per lane) and electrophoretically trans- The authors thank Dr Vale´rio Portela for assistance with ferred onto nitrocellulose membrane in a Bio-Rad wet Blot cultures, Dr L M Sanford for the gift of oestradiol antibody Transfer Cell apparatus (transfer buffer: 39 mM glycine, 48 mM and Dr A F Parlow and National Hormone & Peptide Program, Tris-base, 1% SDS, 20% methanol, pH 8.3). After transfer, the NIDDK for bovine FSH. membranes were blocked in 5% non-fat dry milk in TTBS (10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h. Membranes were incubated overnight with the primary antibody (anti-ERK, #9102, 1:2000; anti-phospho-ERK, #9101, 1:1000; anti-AKT, #9272, 1:1000 and anti-phospho-AKT References (Ser473), #9271, 1:1000; Cell Signaling Technology, Danvers, Anteby EY, Natanson-Yaron S, Greenfield C, Goldman-Wohl D, MA, USA) diluted in 5% BSA in TTBS at 4 8C. After washing Haimov-Kochman R, Holzer H & Yagel S 2005 Human placental three times with TTBS, membranes were incubated for 2 h at Hofbauer cells express Sprouty proteins: a possible modulating mecha- nism of villous branching. Placenta 26 476–483. (doi:10.1016/j.placenta. room temperature with 1:10 000 anti-rabbit HRP-conjugated 2004.08.008) IgG (GE Healthcare Canada) diluted in 5% non-fat dry milk in Baird A & Hsueh AJ 1986 Fibroblast growth factor as an intraovarian TTBS. After five washes in TTBS, protein bands were revealed hormone: differential regulation of steroidogenesis by an angiogenic by chemiluminescence (ECL; Millipore, Billerica, MA, USA) factor. Regulatory Peptides 16 243–250. (doi:10.1016/0167- 0115(86)90023-6) and autoradiography. Semiquantitative analysis was performed Berisha B, Schams D, Kosmann M, Amselgruber W & Einspanier R 2000 with NIH Image J Software (Bethesda, MD, USA). Expression and localisation of vascular endothelial growth factor and basic fibroblast growth factor during the final growth of bovine ovarian follicles. Journal of Endocrinology 167 371–382. (doi:10.1677/joe.0. 1670371) E2 assay Berisha B, Sinowatz F & Schams D 2004 Expression and localization of E2 was measured in medium in duplicate by RIA. The fibroblast growth factor (FGF) family members during the final growth of antibody was raised in rams as described previously (Sanford bovine ovarian follicles. Molecular Reproduction and Development 67 3 162–171. (doi:10.1002/mrd.10386) 1987), and the tracer was E2(2,4,6,7,16,17- H; Perkin Elmer, Berisha B, Steffl M, Amselgruber W & Schams D 2006 Changes in fibroblast Montreal, QC, Canada). Free and bound tracers were growth factor 2 and its receptors in bovine follicles before and after separated by incubation with dextran–charcoal. This antibody GnRH application and after ovulation. Reproduction 131 319–329. crossreacted with oestrone (15%) but not with other steroids (doi:10.1530/rep.1.00798) Bottcher RT & Niehrs C 2005 Fibroblast growth factor signaling during early tested. The intra-assay coefficient of variation of the single vertebrate development. Endocrine Reviews 26 63–77. (doi:10.1210/er. assay used was !7%, and the sensitivity of the assay was 2003-0040) 0.70 ng/ml medium. Buratini J Jr, Glapinski VF, Giometti IC, Teixeira AB, Costa IB, Avellar MC, Barros CM & Price CA 2005 Expression of fibroblast growth factor-8 and its cognate receptors, fibroblast growth factor receptor (FGFR)-3c and-4, in fetal bovine preantral follicles. Molecular Reproduction and Statistical analysis Development 70 255–261. (doi:10.1002/mrd.20205) All statistical analyses were performed with JMP Software (SAS Buratini J Jr, Pinto MGL, Castilho AC, Amorim RL, Giometti IC, Portela VM, Nicola ES & Price CA 2007 Expression and function of Institute, Cary, NC, USA). Data were transformed to logarithms fibroblast growth factor 10 and its receptor, fibroblast growth factor if they were not normally distributed (Shapiro–Wilk test). receptor 2B, in bovine follicles. Biology of Reproduction 77 743–750. ANOVA was used to test the main effects of treatments, (doi:10.1095/biolreprod.107.062273) Cabrita MA & Christofori G 2008 Sprouty proteins, masterminds of and culture replicate was included as a random variable in receptor tyrosine kinase signaling. Angiogenesis 11 53–62. (doi:10.1007/ the F-test. Differences between means were tested with the s10456-008-9089-1) Tukey–Kramer honestly significant difference (HSD) test. Cao M, Nicola E, Portela VM & Price CA 2006 Regulation of serine protease The data are presented as least square meansGS.E.M. inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro. Matrix Biology 25 342–354. (doi:10. 1016/j.matbio.2006.05.005) Cosgrave N, Hill ADK & Young LS 2006 Growth factor-dependent Declaration of interest regulation of survivin by c-myc in human breast cancer. Journal of Molecular Endocrinology 37 377–390. (doi:10.1677/jme.1.02118) The authors declare that there is no conflict of interest that Dailey L, Ambrosetti D, Mansukhani A & Basilico C 2005 Mechanisms could be perceived as prejudicing the impartiality of the underlying differential responses to FGF signaling. Cytokine & Growth research reported. Factor Reviews 16 233–247. (doi:10.1016/j.cytogfr.2005.01.007) Eloy-Trinquet S, Wang H, Edom-Vovard F & Duprez D 2009 Fgf signaling components are associated with muscles and tendons during limb development. Developmental Dynamics 238 1195–1206. (doi:10.1002/ dvdy.21946) Funding Firnberg N & Neubuser A 2002 FGF signaling regulates expression of Tbx2, Erm, Pea3, and Pax3 in the early nasal region. Developmental Biology Z Jiang was supported by a fellowship from the Chinese 247 237–250. (doi:10.1006/dbio.2002.0696) Scholarship Council. The work was supported by a Discovery Gasperin BG, Ferreira R, Rovani MT, Santos JT, Buratini J, Price CA & Grant from the Natural Sciences and Engineering Research Goncalves PB 2012 FGF10 inhibits dominant follicle growth and estradiol secretion in vivo in cattle. Reproduction 143 815–823. Council, Canada. (doi:10.1530/REP-11-0483) www.reproduction-online.org Reproduction (2012) 144 625–632

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