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Departamento de Fisiologia3, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, CEP 18618-000, Brazil
Departamento de Reprodução Animal4 and Departamento de Clínica Veterinária,5 Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Botucatu, São Paulo, CEP 18618-000, Brazil
Centre de Recherche en Reproduction Animale,6 Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
ABSTRACT
Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.
fibroblast growth factors, follicle, granulosa cells, growth factors, theca cells
Ovarian follicle growth is orchestrated by complex interactions between the three main cell types within the follicle: theca cells, granulosa cells, and the oocyte. There is considerable evidence to indicate that members of the transforming growth factor ß superfamily are mediators of paracrine signaling within the follicle [1]. For example, growth/differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are produced by oocytes and affect bovine granulosa cell function [2–6], while BMP4 and BMP7 are theca proteins that alter bovine granulosa cell function [7, 8]. Fibroblast growth factors (FGFs) are also potential paracrine signaling molecules. In the bovine ovary, the FGF7 gene is expressed principally or exclusively in theca cells, although expression appears not be regulated during development of nonatretic follicles [9, 10]. The receptor for FGF7 is the ‘b’ splice variant of the FGFR2 gene [11], referred to herein as FGFR2B, which is expressed predominantly in granulosa cells and is upregulated during follicle development [9, 10].
Another FGF that activates FGFR2B is FGF10, which is structurally similar to FGF7 [12, 13]. In mice, FGF10 is essential for normal development of lungs, limbs, teeth, mammary buds, stomach and heart [14–18]. We hypothesized that FGF10 signaling to FGFR2B plays a role in follicle development, although nothing is known about the expression of FGF10 in the ovary or the biological role of the protein. The first objective of the present study was to examine the developmental and spatial patterns of FGF10 expression in the bovine follicle. We also measured FGF7 mRNA to determine if there were any differences in the expression patterns of these two genes. As FGFR2B gene expression changes during follicle development, the second objective was to determine whether FGFR2B expression in granulosa cells is regulated by FSH or IGF1. Lastly, we investigated whether FGF10 acts upon nonluteinizing granulosa cells in serum-free culture.
A variety of tissues was collected from fetal and adult cows. Adult tissues and whole fetuses were obtained from an abattoir located near the Sao Paulo State University campus in Botucatu and transported to the laboratory in saline on ice. Samples were homogenized in TRIzol (Invitrogen), and stored at –70°C until RNA extraction.
Preantral follicles were harvested from pairs of ovaries obtained from individual fetuses using a Tissue Chopper (Mickle Laboratory Engineering Co.), as described previously [19]. The resulting follicle suspension was examined under an inverted microscope and primordial, primary, and secondary follicles without obvious stroma investment were separated with a capillary pipette [20]. Once isolated, the preantral follicles were pooled within each class (primordial, primary, and secondary) for each fetus, homogenized in 500 µl TRIzol with a Polytron, and stored at –70°C. Total RNA was extracted according to the TRIzol protocol.
Antral follicles were obtained from the ovaries of adult cows. Follicles of diameter 
5 mm were dissected from the ovaries, and follicular fluid was aspirated, centrifuged, and frozen for progesterone and estradiol assays. The antral cavity was flushed repeatedly with cold saline, and granulosa cells were recovered by centrifugation at 1200 x g for 1 min and pooled with the follicular fluid pellet. The remaining granulosa cells adhering to the follicle wall were removed by gently scraping with a blunt Pasteur pipette, and the theca layer was removed with a forceps and washed in saline by repeated passages through a 1-ml syringe. The samples were collected in TRIzol and homogenized with a Polytron. Total RNA was extracted immediately according to the TRIzol protocol.
Follicles were classed according to estradiol:progesterone (E:P) ratios of >1, 1–0.01, and <0.01 [21], which we defined as healthy, transitional, and highly atretic, respectively [22]. Follicles were subgrouped by size (5–7 mm, 8–10 mm, and >10 mm in diameter) and by follicular fluid estradiol concentration (<5, 5–20, >20–100, and >100 ng/ml [23]). Cross-contamination of theca and granulosa cells was tested by PCR detection of the mRNAs that encode cytochrome P450 aromatase (CYP19A1) and 17
-hydroxylase (CYP17A1) in each sample, as described previously [24]. The detection of CYP19A1 amplicons in theca samples or of CYP17A1 amplicons in granulosa samples indicated cross-contamination, and these samples were discarded.
Cumulus-oocyte-complexes were aspirated from antral follicles, and cumulus cells were removed from the oocyte by vortexing. Pools of 50 oocytes were collected, and RNA was extracted with the RNeasy kit (Qiagen).
Granulosa cell culturing was performed as described [25], with modifications [26]. All materials were obtained from Invitrogen Life Technologies, except where otherwise stated. Follicles (
5 mm in diameter) were dissected from slaughterhouse ovaries, and those with obvious signs of atresia (i.e., avascular theca, debris in the antrum) were discarded. Cells were collected by repeatedly passing the follicle wall through a pipette, washing twice by centrifugation at 980 x g for 20 min each, and suspension in -MEM that contained Hepes (20 mM), sodium bicarbonate (10 mM), sodium selenite (4 ng/ml), BSA (0.1%; Sigma-Aldrich), penicillin (100 IU/ml), streptomycin (100 µg/ml), transferrin (2.5 µg/ml), nonessential amino acid mix (1.1 mM), androstenedione (10–7 M at start of culture, and 10–6 M at each medium change), and insulin (10 ng/ml). Cell viability was estimated by 0.4% Trypan Blue staining. Cells were seeded into 24-well tissue culture plates (Corning) at a density of 106/well in 1 ml of medium. The cultures were maintained at 37°C in 5% CO2 in air for 6 days, with 700 µl of medium being replaced every 2 days.
To determine the regulation of FGFR2B expression, cells were stimulated with FSH (AFP-5332B, NIDDK) or IGF1 analog (LR3; Sigma-Aldrich) at the doses listed in Results. At the end of the culture period, cells were collected in TRIzol and stored at –70°C until RNA extraction. To determine the effect of FGF10 on granulosa cells, cells were cultured with FSH (1 ng/ml) and graded doses of recombinant human FGF10 (PeproTech). On Day 6 of culture, the medium was removed for steroid assay and the cells were lyzed with 100 µl of 1 N NaOH for 2 h followed by neutralization with 100 µl of 1 N HCl for total cell protein measurement with the Bradford protein assay (Bio-Rad). Cell proliferation was measured with the MTT assay (R&D Systems). Experiments were performed on three independent culture replicates.
Reverse Transcription-Polymerase Chain Reaction
The primer sequences for FGF7 and FGFR2B were as published and validated for bovine granulosa and theca cells [10, 11]. The primer sequences for FGF10 were those used previously in sheep [27]. The primers for housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH; for follicle samples) and histone 2 H2aa1 (HIST2H2AA1; for cell culture samples) have been described previously [24]. All of the primers spanned at least one intron and all of the PCR reactions produced amplicons of the expected size, demonstrating the absence of contamination by genomic DNA.
Expression of FGF7 and FGF10 was surveyed in fetal and adult bovine tissues by RT-PCR. Total RNA (1 µg) was incubated with DNase I (Invitrogen), and then reverse-transcribed with SuperScript II (Invitrogen) and oligo(dT) primer. PCR was performed with 1 µl cDNA in a PCR master mix that contained 1.6 U Taq DNA polymerase (Invitrogen), 0.4 µM primers, 0.2 mM dNTPs, and 1.5 mM MgCl2 in a total volume of 25 µl. Samples were denatured for 3 min at 94°C, followed by 40 cycles of denaturing at 94°C for 45 sec, annealing at 60°C (for FGF10 and GAPDH) or 65°C (for FGF7) for 45 sec, and extension at 70°C for 1 min.
Expression of FGF10 in isolated granulosa and theca cells was measured by semiquantitative RT-PCR, as described above but in a duplex reaction with GAPDH primers (0.06 µM) for 30 cycles. FGF7 mRNA was measured in simplex reactions with 32 cycles for FGF7 and 24 cycles for GAPDH. Semiquantitative RT-PCR was validated by choosing the number of PCR cycles and amount of RNA within the linear range of the amplification curve.
All PCR reactions were performed with positive (fetal lung) and negative (water replacing cDNA) controls. PCR products were separated on 1.5% agarose gels and stained with ethidium bromide, and the bands were quantified by densitometry (Image Gauge; Fuji Photo Film Co.). The authenticity of the amplified FGF10 cDNA was verified by sequencing the amplicons from bovine oocyte and fetal lung samples; the bovine sequence (GenBank accession no. AY183659) is 98% homologous with the published sheep sequence (NM_001009230).
The RNA yield from oocytes was too low to be quantified accurately by spectrometry, so 8-µl aliquots of RNA were reverse-transcribed with SuperScript III (Invitrogen). PCR was performed with 1 µl cDNA and 1 U Platinum Taq (Invitrogen) for 40 cycles (for FGF7 and FGF10) or 30 cycles (for GAPDH).
For the analysis of FGFR2B expression in cultured granulosa cells, RNA was reverse-transcribed with Omniscript (Qiagen), and semiquantitative RT-PCR was performed as described for freshly harvested granulosa cells, with the exceptions that the PCR was performed with Platinum Taq for 38 cycles and that HIST2H2AA1 was used as the housekeeping gene (30 cycles).
Bovine ovaries were collected from an abattoir, bisected, and fixed in paraformaldehyde. Fixed tissues were embedded in paraffin, and 3-µm sections were placed on poly-L-lysine-coated slides. Sections were deparaffinized in xylene twice for 30 min, and hydrated in successive 5-min washes in 95% and 85% ethanol. Formaldehyde was washed from the sections by incubation with 10% ammonium hydroxide in ethanol for 10 min, followed by washing in distilled water. Antigen retrieval was achieved by incubating the sections in 10 mM EDTA (pH 8.0) at 96°C for 25 min. Endogenous peroxidase was quenched by incubation in hydrogen peroxide for 20 min, followed by two rinses for 5 min in distilled water and one rinse in 0.5 M Tris (pH 7.4).
Tissue sections were blocked in 5% BSA in Tris for 1 h at room temperature in a humidified chamber, and then incubated with polyclonal anti-FGF10 antibody (H-121, 1:200 or 1 µg/ml; Santa Cruz Biotechnology) overnight at 4°C. Slides were washed twice for 5 min in Tris, and then incubated with horseradish peroxidase-conjugated secondary antibody (EnVision Dual Link System; DakoCytomation). Immunostaining was developed with liquid DAB (DakoCytomation). Negative controls involved omission of the primary antibody from the procedure or preadsorption of the primary antibody (1 µg/ml) with human recombinant FGF10 (2 µg/ml) overnight at 4°C before application to the slides.
Estradiol and progesterone were assayed in follicular fluids using iodinated tracers and the antibodies furnished in the Third Generation Estradiol RIA and Progesterone RIA kit (Diagnostic Systems Laboratories Inc.), respectively, with a revised protocol [24]. The standard curves and samples were diluted in PBS-gelatin. The intraassay and interassay coefficients of variation were 7.4% and 13.5%, respectively, for estradiol, and 6.8% and 7.0%, respectively, for progesterone. The sensitivities of the assays were 0.3 ng/ml for estradiol and 0.2 ng/ml for progesterone.
Estradiol was measured in culture medium with the RIA as described but without solvent extraction [28], and progesterone was measured as described [29]. All samples were run in a single assay for each steroid, with intra-assay coefficients of variation of 8.5% and 7.2% for estradiol and progesterone, respectively. The sensitivities of these assays were 10 pg and 4 pg per tube for estradiol and progesterone, which are equivalent to 0.3 and 20 ng/µg protein, respectively.
Target gene mRNA abundance is expressed relative to the level of GAPDH (for follicles) or HIST2H2AA1 (granulosa cell cultures) mRNA. Estradiol secretion in vitro was corrected for cell number by expression per unit mass of total cell protein. The data were transformed to logarithms if not normally distributed. ANOVA was used to test for the main effects of follicle class, size, and estradiol concentration on FGF7 and FGF10 mRNA levels in theca cells, the effects of FSH and IGF1 on FGFR2B mRNA levels in granulosa cells in vitro, and the effect of FGF10 protein on granulosa cell estradiol secretion and proliferation in vitro. For the cell culture experiments, culture replicate was included as a random effect in the F-test; owing to the significant effect of replicate on estradiol secretion, the data are expressed relative to the control (no FGF10). Means comparisons were performed with the Tukey-Kramer HSD test. Data are presented as means ± SEM. Analyses were performed with the JMP software (SAS Institute, Cary, NC).
FGF7 and FGF10 were widely expressed in two bovine fetuses (Fig. 1). The only tissue examined that lacked expression of FGF10 in both fetuses was the liver, although one fetus also failed to show expression in the brain (Fig. 1). Expression in adult tissues followed a similar distribution but was less readily detectable (Fig. 1). In the ovary, FGF7 mRNA was evident in the theca cells but not in the granulosa cells or oocytes. FGF10 mRNA was detected in theca cells and oocytes but not in granulosa cells (Fig. 2). FGF10 and FGF7 mRNAs were expressed in primordial, primary, and secondary follicles, all of which showed robust GAPDH mRNA expression (Fig. 3).
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Immunohistochemistry revealed the presence of FGF10 protein in the oocytes of the preantral and antral follicles, as well as in the theca and granulosa cell layers of the antral follicles (Fig. 4). A low level of staining was observed in stromal tissues, including blood vessels. No staining was observed in the absence of primary antibody or with primary antibody that was preadsorbed with FGF10 (Fig. 4).
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Developmental regulation of FGF7 and FGF10 expression was assessed by measuring the mRNA levels in the theca cells of follicles classified according to size and follicular fluid steroid concentration. Estradiol concentrations were higher in healthy follicles compared to transitional follicles (P < 0.001), which in turn had higher levels than the highly atretic follicles (Table 1; P < 0.001). Although the progesterone concentrations were not different between healthy and transitional follicles, they were significantly higher in highly atretic follicles (P < 0.001). Mean follicle diameter did not differ between the groups (Table 1). ANOVA indicated that FGF7 expression was not significantly affected by the E:P ratio (Table 1), estradiol concentration or diameter. However, FGF10 expression was significantly affected by the E:P ratio and estradiol concentration. Consequently, follicles were classified by estradiol concentration within the E:P groups. FGF10 mRNA abundance was highest in the transitional follicles that contained <5 ng/ml estradiol and lower in the follicles that contained 5–20 ng/ml or 20–100 ng/ml estradiol. No transitional follicles contained >100 ng/ml estradiol. Healthy follicles that contained 20–100 ng/ml estradiol expressed levels of FGF10 mRNA that were similar to those of the transitional follicles in the same estradiol group, and expressed more FGF10 than healthy follicles that contained >100 ng/ml estradiol. No healthy follicles contained <20 ng/ml estradiol. The lowest FGF10 expression was observed in highly atretic follicles (Table 1, Fig. 5A), none of which contained >5ng/ml estradiol. Within a follicle group, there was no effect of diameter on FGF10 expression (Fig. 5B).
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The gene that encodes FGFR2B, which is the major receptor for FGF7 and FGF10, was expressed in cultured granulosa cells. There were significant and opposing effects of FSH and IGF1 on FGFR2B expression, i.e., FSH increased (P < 0.01) and IGF1 decreased FGFR2B mRNA abundance in cells that were cultured for 6 days in serum-free medium (P < 0.05) (Fig. 6).
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To assess the role of FGF10 signaling, FSH-stimulated granulosa cells were cultured in the presence of graded doses of FGF10. Estradiol secretion was inhibited (P < 0.05; Fig. 7) by FGF10 in a dose-dependent manner, whereas cell proliferation was not affected (Fig. 7).
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There is increasing evidence for FGF participation in follicle development, including roles for FGF2 [30] and FGF7 [9]. In the present report, we present four lines of evidence that FGF10 is also involved in follicle growth. First, both the mRNA and protein encoded by the FGF10 gene were detected in oocytes and theca cells. Second, theca expression of FGF10 changed with follicle health. Third, granulosa cell expression of FGFR2B, which is the major receptor for FGF10, was regulated by FSH, and fourth, FGF10 inhibited estradiol secretion from cultured granulosa cells.
We have demonstrated for the first time that the FGF10 gene is expressed in the ovary. FGF7 and FGF10 mRNAs were detected in theca cells, consistent with the mesenchymal origin of these growth factors [12, 31]. The expression of FGF10 was confirmed by localizing FGF10 protein to the theca layer. This protein was also detected in the granulosa layer, whereas the associated mRNA was not. This discrepancy may be explained by the internalization of FGF10 after binding to its receptor, which is expressed by granulosa cells ([9, 10] and present study). This is supported by evidence for receptor-mediated internalization of exogenous FGF1 and FGF2 in several cell lines [32, 33], and of most relevance to the present work is the internalization of FGF7 after binding to FGFR2B in NIH3T3 fibroblasts [34, 35]. FGF10 detection in the granulosa appears to be specific, as preincubation of the anti-FGF10 antibody with FGF10 abolished staining, although we cannot rule out the possibility that this antibody cross-reacts with other bovine FGFs.
Of great interest is the presence of FGF10 protein in the oocytes of preantral and antral follicles and the detection of FGF10 mRNA in isolated oocyte pools and preantral follicles. Thus, this member of the FGF family joins FGF2 [36] and FGF8 [24, 37] in the group of potential oocyte-derived signaling molecules. In contrast, we show in the present study that FGF7 is expressed in bovine preantral follicles but not in oocytes. Although the preantral follicles used in the present study were carefully selected [20], we cannot discount the possibility that FGF7 mRNA arises from contamination with stromal cells, as suggested by the presence of FGF7 protein in rodent ovarian stroma but not in oocytes [38]. It has been proposed that stromal cell FGF7 signals via FGFR2B to promote the activation of primordial follicles in rats [38]. In light of the considerable FGF10 immunostaining detected in the oocytes of the preantral follicles, we propose that oocyte-derived FGF10 promotes follicle activation in cattle. This is difficult to test directly, as a high percentage of bovine primordial follicles spontaneously activate in vitro [39, 40], which precludes the study of factors that stimulate primordial follicle transition.
The expression of FGF10 in theca cells was developmentally regulated. We classified the follicles with E:P ratios >1 as healthy and those with E:P ratios <0.01 as highly atretic, which are widely recognized criteria [21]. We defined those follicles with E:P ratios of 0.01–1.0 as transitional, based on the report of Grimes and Ireland [22], who described these follicles as progressing from the nonatretic to atretic stage. However, since smaller growing follicles also have E:P ratios of 0.01–1.0 [41, 42], the transitional follicle group probably includes both growing and early atretic follicles. For both transitional and healthy follicles, FGF10 mRNA abundance decreased as the estradiol concentration increased, which suggests that FGF10 plays a role during growth of the smaller, less-estrogenic follicles. Highly atretic follicles displayed a marked reduction in FGF10 expression, which suggests that this gene is downregulated in theca cells during atresia. This is in contrast to the expression of FGF7, which was not altered by the E:P ratio or estradiol concentration in the follicles ([10], and present study), although it has been reported that FGF7 expression is higher in follicles of >10 mm in diameter than in smaller follicles [9]. The pattern of FGF10 expression in theca cells also differed from the expression patterns observed for FGF1, which did not change with estradiol concentration in bovine antral follicles, and FGF2, which increased with estradiol concentration [10]. Although somewhat speculative, these data suggest that different FGFs act at different times during follicle development, with FGF10 having a greater role during the early-mid phase of antral follicle growth.
FGF receptor expression is also regulated, as granulosa cell FGFR2B expression increased with increasing estradiol concentrations in bovine follicles [10]. In the present study, we show that FGFR2B mRNA in granulosa cells is upregulated by FSH in a manner similar to that described for granulosa FGFR3C [24]. Neither of these receptors were upregulated by IGF1 ([24] and present study), and indeed we show that IGF1 suppresses FGFR2B expression. The progressive increase of FGFR2B expression with follicle growth in vivo [10] is similar to the increases in other FSH-responsive genes, such CYP19A1 [43, 44], suggesting an important role for this receptor during follicle growth.
To determine the potential biological importance of FGF10 signaling for granulosa cells, we tested the effect of FGF10 on estradiol secretion and cell proliferation in nonluteinizing bovine granulosa cells in serum-free culture. This culture system represents the most physiologically relevant model of granulosa cell differentiation available in ruminants, as steroidogenic enzymes that are upregulated or induced during granulosa cell differentiation in vivo are also upregulated or induced in vitro, and there is no upregulation of the luteinization marker, steroidogenic acute regulatory protein [45]. In this cell model, FGF10 significantly inhibited estradiol secretion without affecting cell proliferation. This is consistent with FGF7 inhibition of aromatase activity in bovine granulosa cells [9]. Therefore, paracrine control of estradiol secretion appears to be a balance between inhibitory (GDF9 [5]; FGF10, present study) and stimulatory (BMP4 and BMP7 [7, 8]) factors.
FGF10 did not affect cell proliferation in the present study, although FGF7 has been reported to stimulate granulosa cell proliferation [46]. The effects of some growth factors on ruminant granulosa cell proliferation remain controversial; whereas McNatty et al. [6] have reported no effect of ovine GDF9 on bovine granulosa cell proliferation, Spicer et al. [5] have reported that rat GDF9 stimulates bovine granulosa cell proliferation. These discrepancies may be related to the source (species) of the peptide hormones or the method used to measure cell proliferation (thymidine incorporation vs. MTT assay).
In summary, the present report provides new evidence for paracrine FGF10 signaling in bovine follicles. Based on the patterns of gene expression and actions of FGF10 discussed above, a possible model of FGF10 action can be formulated as follows. Theca cells from early antral follicles express the FGF10 gene. The rise in FSH that precedes a wave of follicle growth [47, 48] increases FGFR2B expression in granulosa cells, which enhances the response to secreted FGF10. The action of FGF10 at this stage is to inhibit the differentiation of granulosa cells, as reflected by limited estradiol secretion, which is a major marker of differentiation [43, 44]. As follicle development proceeds, there is a progressive decrease in theca FGF10 secretion, which allows increased estradiol secretion and granulosa cell differentiation.
ACKNOWLEDGMENTS
We thank Dr. M.C.W. Avellar (Universidade Federal de São Paulo, Escola Paulista de Medicina) for sequencing the FGF10 PCR product, Dr. P.R. Ramos (Universidade Estadual Paulista, Botucatu) for access to gel documentation equipment, and Drs. A.B. Teixeira, I.B. Costa, and V.F. Glapinski for technical assistance.
FOOTNOTES
1Supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil, and the Natural Sciences and Engineering Research Council of Canada. 
Correspondence: 2FAX: 55 14 38116251; e-mail: buratini{at}ibb.unesp.br
Received: 15 April 2007.
First decision: 5 May 2007.
Accepted: 7 June 2007.
REFERENCES
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