Fibroblast Growth Factors 1 and 2 in the Primate Uterus

^a Section on Women's Health, Developmental Endocrinology Branch, NICHD, NIH, Bethesda, Maryland 20892

	ABSTRACT

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Fibroblast growth factors (FGF) 1 and 2 are paracrine effectors of proliferation and angiogenesis in many tissues. To elucidate potential roles for these growth factors in uterine plasticity, we used in situ hybridization histochemistry to identify the cellular sources of FGF-1 and -2 production, and immunohistochemistry to identify the cellular and extracellular deposition sites of the peptides in the primate uterus. To evaluate the effects of estradiol on uterine FGFs, uteri from ovariectomized rhesus monkeys treated with estradiol- or vehicle-containing pellets were investigated. FGF-1 and -2 mRNAs were both expressed in uterine epithelial and myometrial cells. Quantitative comparison of their mRNA levels using computerized grain counting showed no significant difference between estradiol- and vehicle-treated animals.

FGF-1 immunoreactivity was detected in scattered epithelial, vascular, and myometrial cells in the vehicle-treated animals but found to be significantly more intense and widespread in estradiol-treated animals. In both conditions, FGF-1 immunostaining was predominantly nuclear. FGF-2 immunoreactivity was concentrated extracellularly in the basal lamina of both glandular and surface epithelium and was abundant and diffusely distributed within myometrial and vascular cells in both cytoplasm and nucleus. There was no apparent difference in the pattern or intensity of FGF-2 immunostaining related to estradiol treatment. These data demonstrate that major uterine cell types synthesize both FGF-1 and -2, and that the two peptides are differentially localized in uterine cellular and extracellular compartments and differentially sensitive to regulation by estradiol.

	INTRODUCTION

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The fibroblast growth factor (FGF) family consists of multiple structurally related peptides, the best characterized of which are acidic and basic FGFs (FGF-1 and FGF-2, respectively). FGF-1 and FGF-2 are heparin-binding polypeptides whose functions include regulation of proliferation, differentiation, and angiogenesis [1]. The recognized functions of FGFs in tissue plasticity are potentially important for the cyclic growth, differentiation, and neovascularization that characterize uterine responses to sex steroids. FGF-2 peptide or mRNA has been identified in murine, rabbit, and human endometria [2–7]. FGF-1 has not been as widely studied, but FGF-1-like activity has been isolated from porcine uterus [8].

The cellular source of FGF production and cellular or extracellular sites of FGF peptide localization in the primate uterus have not been comparatively analyzed. To elucidate the potential cellular interactions involving FGFs in the primate uterus, we have used histological techniques to identify the sources of uterine FGF-1 and -2 production by localizing their respective mRNAs using in situ hybridization. Furthermore, we have identified their cellular targets or storage sites by immunohistochemically localizing the FGF-1 and -2 peptides in the uterus. To assess the potential regulation of FGF synthesis by estradiol, we conducted these studies in parallel, using uterine tissues from ovariectomized monkeys that received pellets containing vehicle or estradiol.

	MATERIALS AND METHODS

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Animals

Female rhesus monkeys (Macacca mulatta) 6–13 yr of age from the National Institutes of Health colony in Poolesville, MD, were used in accordance with a protocol approved by the National Institute of Child Health and Human Development Animal Care and Use Committee. Ovariectomies were performed on nine monkeys under ketamine anesthesia (Fort Dodge Lab., Fort Dodge, IA) via a midventral laparotomy in the follicular phase of the menstrual cycle. Two weeks after surgery, animals were randomly assigned to three groups. One group (control, n = 8) had vehicle pellets (Innovative Research of America, Toledo, OH) inserted s.c. under ketamine anesthesia between their shoulder blades, while the others received sustained-release estradiol-17ß pellets (20 mg) for 3 days (n = 5) or 15 days (n = 3). After 3 or 15 days, the animals were sedated with ketamine and then killed with pentobarbital (65 mg/kg). Uteri were removed, snap-frozen on dry ice, and stored at -70°C. Serial sections of 10-µm thickness were cut at -15°C and thaw-mounted onto poly-L-lysine-coated slides for in situ hybridization and immunocytochemistry.

Immunohistochemistry

For immunohistochemistry, slides were fixed in acetone at 20°C for 15 min and air-dried for 15 min. After two washes in PBS with 0.05% Tween and one 5-min wash with PBS alone, sections were blocked with the avidin-biotin complex (Vector Laboratories, Burlingame, CA) for 30 min at 25°C. Thereafter, tissue sections were washed in PBS with 0.05% Tween followed by PBS alone. Primary antibodies were diluted in PBS + 10% horse serum and applied to the sections for 1 h at 25°C in a humidified chamber. For each anti-FGF antibody, the dilution that yielded optimal specific staining was determined in pilot experiments. The following primary antibodies were used: mouse monoclonal anti-bovine FGF-2 (10 µg/ml) and mouse monoclonal anti-human FGF-1 (10 µg/ml; both from Upstate Biotechnology Inc., Lake Placid, NY). After two washes in PBS with 2% horse serum, a biotinylated secondary anti-mouse antibody (1:100 dilution; Vector Laboratories) was applied for 45 min. After two washes for 10 min each in PBS with 2% horse serum and once in PBS alone, the ABC Elite solution (ABC Elite Kit; Vector Laboratories) was applied for 45 min in a humidified chamber. The tissue sections were then washed twice for 5 min in PBS, and the antigen-antibody complex was visualized by incubation with freshly prepared 3,3'-diaminobenzidine (DAB substrate kit; Vector Laboratories) and counterstained with methyl green dye. Control slides were carried through the same procedure, but BSA rather than primary antibody was used.

Western Blot Analysis for FGF-1 and FGF-2 Cross-Reactivity

To investigate potential cross-reactivity of FGF-1 and FGF-2 with our anti-FGF antibodies, immunoblot analysis using recombinant FGF peptides (Wako Chemicals USA, Inc., Richmond, VA) was performed. Recombinant human FGF-1, a 140-amino acid peptide of approximately 15.5 kDa, and recombinant human FGF-2, a 154-amino acid peptide of approximately 17 kDa, were subjected to SDS-PAGE in 10% gradient gels and were then electrophoretically transferred to nitrocellulose membranes. After blocking of nonspecific protein-binding sites with 3% dry milk in TBS, the nitrocellulose membrane was incubated with monoclonal anti-human FGF-1 (10 µg/ml) and monoclonal anti-bovine FGF-2 (10 µg/ml) overnight. After incubation with horseradish peroxidase-linked secondary antibodies, the protein bands were detected by enhanced chemiluminescence (ECL) detection reagents (Amersham, Cleveland, OH) and visualized on film.

RNA Probes

FGF-1 and FGF-2 complete cDNA sequences were obtained from American Type Culture Collection (ATCC #63347 and 63348, respectively). A 468-basepair (bp) complete cDNA sequence encoding mouse FGF-1 and a 465-bp mouse FGF-2 were subcloned into pBluescript SK, and the plasmids were linearized with Xba I for use as templates to synthesize antisense probes [9]. The FGF-1 and FGF-2 cDNA probes had 57.5% sequence homology [10]. A human insulin-like growth factor 1 (IGF-1) cDNA with similar length (442 bp) was subcloned into the plasmid vector pGEM-3 and transcribed in a reverse direction for use as a control sense probe [11]. This probe was used for sense hybridization of parallel tissue sections because the FGF sequences are present in both sense and antisense forms in many tissues [12].

The synthesis of ³⁵S-labeled cRNA probes has been previously described in detail [13]. Briefly, 1.0-µg DNA template, 0.1 mCi [³⁵S]CTP, and 0.1 mCi [³⁵S]UTP were added to a microcentrifuge tube and dried down in a speed vacuum, after which 2 µl nucleoside triphosphate mix, 1 µl dithiothreitol (DTT), 2 µl specific polymerase buffer, 1 µl RNA polymerase, 1 µl RNasin, and 3 µl sterile water were added. The mixture was then incubated for 60 min at 42°C. RNase-free DNase was then added and incubated for 10 min at 37°C to digest the DNA template. After column separation (Bio-Spin 6 Column; Bio-Rad, Richmond, CA), the probes were precipitated, alkaline-hydrolysed [13], and resuspended in 50 µl Tris (10 mM)-DTT (20 mM). The probes were then used for the hybridization experiments.

In Situ Hybridization

Before hybridization, sections were warmed to 25°C, fixed in 4% formaldehyde, and soaked for 10 min in 0.25% acetic anhydride/0.1 M triethanolamine hydrochloride/0.9% NaCl. The tissue sections were then dehydrated through an ethanol series, rehydrated, and air-dried. The ³⁵S-labeled probes (10⁷ disintegrations per min (d.p.m.)/ml or approximately 50 ng/ml) were added to hybridization buffer composed of 50% formamide, 0.3 M NaCl, 20 mM Tris HCl (pH 8), 5 mM EDTA, 500 µg tRNA/ml, 10% dextran sulfate, 10 mM DTT, and 0.02% of BSA, ficoll, and polyvinylpyrrolidone. The ³⁵S-labeled probe in hybridization buffer was then applied to the tissue sections and coverslipped. Parallel sections were hybridized to a ³⁵S-labeled IGF-1 receptor sense probe of length and base composition comparable to those of the FGF sequences. FGF sense probes could not be used to establish nonspecific signal since these genes are transcribed in both directions. The slides were then incubated in humidified chambers overnight (14 h) at 55°C and washed several times in 4-strength saline sodium citrate (SSC; single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) to remove cover slips and hybridization buffer. The sections were then dehydrated and immersed in 0.3 M NaCl, 50% formamide, 20 mM Tris HCl, and 1 mM EDTA buffer at 60°C for 15 min. After dehydration, sections were treated with RNase A (20 µg/ml) for 30 min at room temperature, then washed for 15 min in 0.1-strength SSC at 50°C. Slides were air-dried, apposed to Hyperfilm-beta Max (Amersham) for 7 days, and then dipped in Kodak NTB2 nuclear emulsion (Eastman Kodak, Rochester, NY), stored with desiccant at 4°C for 20 days, developed, and stained with Mayer's hematoxylin and eosin for microscopic evaluation.

Quantitation

FGF-1 and FGF-2 immunostaining were compared in control versus estradiol-treated groups. Tissue sections were studied microscopically at a magnification of x400, and immunostaining was evaluated by a semi-quantitative scoring system. The intensity of the immunoreactive signal for each cell type was assigned a score of 1–4, with 4 being the most intense. Two sections were evaluated for each animal, to obtain a mean score for each cell type in each animal. The results were not significantly different in the 3-day versus the 15-day estradiol-treated groups when compared by ANOVA, so these data were pooled for comparison to control values. The results for each group were compared by the Mann-Whitney U analysis (Statview II; Abacus Concepts, Berkeley, CA) as previously described [14].

In situ hybridization with FGF-1 and FGF-2 mRNAs were quantified by image analysis using darkfield illumination on a Leitz DM RX microscope (Leitz Wetzlar GBH, Wetzlar, Germany) connected to a Macintosh Power PC-based computer analysis system. Hybrid signal was measured over in uterine stroma and myometrium. Grains overlying an area of 100 µm² were captured at x400 via a solid-state monochrome video camera, and the data were analyzed using the NIH Image v1.57 software. Background signal obtained in control sense probe hybridized sections was subtracted from totals for each section before further analysis. Data on mRNA levels were compared using ANOVA, and differences between means were evaluated by Fisher's least-significant-difference method (Statview II; Abacus Concepts).

	RESULTS

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Specificity of the Anti-FGF-1 and -FGF-2 Antibodies

Recombinant FGF-1 and -2 peptides were electrophoresed on polyacrylamide gels and transferred to nitrocellulose membranes. The primary anti-FGF-1 and anti-FGF-2 antibodies were used to probe these blots at a concentration 10-fold higher than was used for immunohistochemistry. There was minimal cross-reactivity of the anti-FGF-2 antibody detected after prolonged exposure of the blots and virtually no cross-reactivity of the anti FGF-1 antibody (Fig. 1).

View larger version (83K): [in this window] [in a new window]   FIG. 1. Anti-FGF-1 and -2 antibodies demonstrated marginal cross-reactivity with the homologous peptides. Western blots of recombinant human-1 and FGF-2 peptides (0.3 or 1.0 µg) were probed with anti-FGF-1 (right) and anti-FGF-2 (left) antibodies. The bands at approximately 17, 34, and 50 kDa represent monomers, dimers, and trimers of the FGF peptides.

FGF-1

Immunohistochemical localization techniques demonstrated the presence of FGF-1 within the uterus. In the ovariectomized, vehicle-treated monkey uterus, there were a few cells in both endometrial glands and surface epithelium that demonstrated FGF-1-immunoreactivity, and the stain was exclusively nuclear (Fig. 2A). There was a suggestion of FGF-1 immunoreactivity along the extracellular lumenal border of the epithelium, but this was not very consistently observed. Little staining was seen along the epithelial basal lamina or in the stromal extracellular matrix. There were scattered immunoreactive nonvascular cells in the stroma, and vascular components of the endometrium demonstrated weak FGF-1 immunoreactivity (Fig. 2A). Within the myometrium of the ovariectomized, vehicle-treated animals, there was a patchy weak signal in groups of muscle cells in both the inner circular and outer longitudinal smooth-muscle layers. In myometrial cells, there was a distinct granular nuclear staining pattern (Fig. 2B). FGF-1 immunoreactivity was substantially more intense and widespread in uteri from estradiol-treated animals (Fig. 2, C and D, and Table 1). The cellular distribution and subcellular pattern remained unchanged; however, the number of cells that were clearly FGF-1-positive and the intensity of the nuclear signal were significantly increased in the estradiol group.

View larger version (140K): [in this window] [in a new window]   FIG. 2. Localization of FGF-1 (A–D) and FGF-2 (E and F) immunoreactivity in the primate uterus. A and B demonstrate FGF-1 in the endometrium (A) and myometrium (B) of an ovariectomized and vehicle-treated monkey. There was a positive signal in a few glandular epithelial cells and a very faint positive signal in vascular structures seen at the bottom and left-hand portion of A. The FGF-1 immunostain was very intense in the myometrial vasculature seen in B, and a very faint nuclear signal is seen in the myometrial cells in this micrograph from a representative untreated animal. C and D show enhanced FGF-1 immunoreactivity in the endometrium (C) and myometrium (D) of an estradiol-treated animal. There was an intense granular nuclear localization of FGF-1 in glandular epithelial and myometrial cells that was enhanced by estradiol treatment. FGF-2 immunoreactivity was localized in the basal laminae associated with uterine glands and blood vessels and was present in scattered endometrial cell cytoplasm (E). These cells were most likely tissue-immune or inflammatory cells. FGF-2 was detected diffusely in myometrial fibers and the cytoplasm and nuclei of vascular cells (F). There was no apparent difference in the abundance of FGF immunoreactivity in the surface versus glandular epithelium. Ep, epithelium; G, uterine glands; V, blood vessels. Bar = 15 µm for A–D and 25 µm for E and F.

In situ hybridization histochemistry showed that FGF-1 mRNA was present in surface and glandular epithelium and, less abundantly, in endometrial stroma (Fig. 3A). It was also diffusely localized in the myometrium. The comparison of FGF-1 mRNA uterine signal in vehicle- and estradiol-treated animals suggested increased expression in the endometrial stroma and myometrium, but these changes were not statistically significant in either the 3-day or 15-day estradiol-treated groups compared with controls (data not shown).

View larger version (150K): [in this window] [in a new window]   FIG. 3. Cellular localization of FGF-1 (A and B) and FGF-2 (C and D) mRNAs in the primate uterus. Both mRNAs were selectively concentrated in the glandular epithelium (Ep, A and C) and smooth muscle cells of the myometrium (B and D). Bar = 25 µm.

FGF-2

FGF-2 immunoreactivity demonstrated a distinctive pattern in the primate uterus. FGF-2 immunoreactivity was concentrated in the basal laminae associated with glandular and surface epithelium (Fig. 2E). The epithelial cells themselves, however, consistently lacked immunoreactive FGF-2 (Fig. 2E). In addition, FGF-2 immunoreactivity was not detected within either the uterine lumen or the lumen of individual endometrial glands (Fig. 2E). Basal laminae associated with vascular elements also demonstrated FGF-2 immunoreactivity throughout the uterus (Fig. 2, E and F). Within the myometrium, intense immunoreactive FGF-2 was observed in both inner circular and outer longitudinal smooth muscle layers (Fig. 2F). In contrast to FGF-1, FGF-2 immunoreactivity in the myometrium was consistently cytoplasmic as well as nuclear, with relatively less intensity seen in the nuclear localization. Also in contrast to FGF-1, there was no discernible effect of estradiol on FGF-2 immunoreactivity except for the vascular elements (Table 1).

In situ hybridization showed that FGF-2 mRNA was present in the glandular and surface epithelium and somewhat less abundantly in endometrial stroma (Fig. 3C) and myometrial smooth muscle cells (Fig. 3D). There were no differences, significant or otherwise, between FGF-2 mRNA levels in the vehicle- versus estradiol-treated groups. Parallel control sections were run for determination of nonspecific signal for both the immunohistochemistry and in situ hybridization. In both methods, the nonspecific signal was extremely low (Fig. 4).

View larger version (101K): [in this window] [in a new window]   FIG. 4. Controls for immunohistochemistry (A) and in situ hybridization (B). Background signal for both techniques was very low. Bar = 50 µm.

	DISCUSSION

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This study has shown that both FGF-1 and -2 mRNAs and peptides are present in the primate uterus. In situ hybridization demonstrated that FGF-1 and -2 mRNAs were relatively abundant in uterine epithelial, smooth muscle, and vascular cells and minimally expressed by stromal and connective tissue cells. Interestingly, while the sites of peptide synthesis were the same, the sites of peptide localization, representing cellular and subcellular targeting or sequestration in extracellular matrix, were quite distinct for FGF-1 and -2. FGF-1 immunoreactivity was concentrated in epithelial, vascular, and myometrial cell nuclei in a discrete, granular pattern. FGF-2 immunoreactivity, in contrast, was localized in the extracellular matrix along the basal lamina underlying the endometrial epithelium but was not detected within epithelial cells. FGF-2 was also abundant in smooth muscle cells of the myometrium, where it was detected in both cytoplasm and nucleus in a rather homogeneous distribution. Finally, we did not detect changes in either FGF-1 or 2 mRNA levels related to estradiol treatment, while FGF-1 but not FGF-2 immunoreactivity demonstrated marked augmentation in estradiol-treated animals.

Previous studies have reported FGF-2 expression in murine, rabbit, and human uteri [2–7]. In the mouse, FGF-2 immunoreactivity was found in a distribution similar to that now reported for the rhesus monkey [2]. Likewise, in the mouse uterus there was no apparent relation between FGF-2 immunoreactivity and estradiol status [2]. The accumulation of FGF-2 in the basal lamina beneath the endometrial epithelium has been suggested to play a role in blastocyst implantation [15]. FGFs have potent pro-proliferative and angiogenic activities and hence might be involved in the tissue remodeling and neovascularization that accompanies trophoblast invasion and blastocyst implantation. In support of the possible involvement of FGF-2 in this process, pregnancy is inhibited in rodents immunized against FGF-2 [16]. The present study shows that the nonhuman primate uterus demonstrates a very similar cellular pattern of FGF-2 expression and suggests that it may play a similar role in higher species.

Furthermore, the present study, in comparing the localizations of FGF mRNA and peptides, provides novel insights into potential mechanisms of FGF action in the primate uterus. Both FGFs lack the classical signal peptide sequence needed to direct secretion, and hence a number of alternative mechanisms have been suggested to explain FGF release from cells. For example, release by cell death is possible. Once released from cells, FGFs are associated with extracellular matrix and released by specific enzymes that degrade the insoluble extracellular matrix structure. Regarding FGF-2, the fact that FGF-2 mRNA is localized in uterine epithelial cells while FGF-2 immunoreactivity is concentrated in the adjacent basal lamina indicates that the peptide is produced by epithelial cells, released in some fashion, and stored immediately next to them in the basal lamina. In the myometrium, by way of contrast, both FGF-2 mRNA and immunoreactivity are abundant within myofibrils. One interpretation of this finding is that FGF-2 is synthesized and then stored within smooth muscle cells, potentially acting in an intracrine manner without ever being secreted from the cell of origin. Alternatively, FGF-2 could be secreted and then taken up in a receptor-mediated fashion by the cell of origin or neighboring cells, thus acting in an autocrine or paracrine fashion in the myometrium.

Although an FGF-1-like electrophoretic species was identified in porcine uterus [8], this is, to our knowledge, the first data identifying specific FGF-1 transcripts and immunoreactivity in the uterus of any species, as well as the first description of the cellular localization of uterine FGF-1 synthesis and targeting. FGF-1 immunoreactivity is interesting in that it is quite different from that of FGF-2, implying different roles for the two peptides in uterine biology. FGF-1 immunoreactivity is predominantly nuclear, and very little is detected in the extracellular compartment. Low levels of FGF-1 in the extracellular matrix could escape immunodetection, but the preponderance of FGF-1 is clearly targeted to the nuclei of FGF-1-synthesizing cells. Recent evidence on the nuclear localization of FGF-1 in many cell types indicates that plasma membrane internalized exogenous FGF is translocated into the nucleus [17]. Thus, it is possible that FGF-1 is first released into the extracellular compartment and then taken up in a receptor-mediated fashion and transported to the nucleus. Alternatively, it is possible that FGF-1 is not released from the cell but transported from the cytoplasm to the nucleus in an "intracrine" mode of action. Imamura et al. [18] have demonstrated that nuclear localization of FGF-1 is necessary for mitogenic activity. Given its nuclear localization in uterine epithelial and smooth muscle cells and up-regulation by estradiol, it seems possible that FGF-1 functions as a mediator of estradiol's mitogenic effects upon these cell types. The fact that FGF-1 immunoreactivity was robustly increased while its mRNA levels were not appreciably increased by estradiol suggests that the peptide abundance is regulated at the level of translation or stability.

In summary, this study has provided the first evidence that primate uterine cells synthesize both FGF 1 and FGF 2 and that these peptides are differentially localized within the uterus and differentially sensitive to regulation by estradiol, implying different roles in primate uterus biology.

	FOOTNOTES

 
¹ Correspondence: Carolyn Bondy, Bldg. 10/10N262, 10 Center Dr., NIH, Bethesda, MD 20892. FAX: (301) 402-2922; bondyc{at}cc1.nichd.nih.gov

Accepted: April 13, 1998.

Received: February 20, 1998.

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