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BOR - Papers in Press, published online ahead of print February 5, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.011270
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BIOLOGY OF REPRODUCTION 68, 2314–2321 (2003)
DOI: 10.1095/biolreprod.102.011270
© 2003 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Characterization of Fibroblast Growth Factor Receptors Expressed in Principal Cells in the Initial Segment of the Rat Epididymis1

Jennifer L. Kirby, Ling Yang, Jacquelyn C. Labus, and Barry T. Hinton2

Department of Cell Biology, University of Virginia Health System, Charlottesville, Virginia 22908


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies from our laboratory support a model in which growth factors produced in the testis reach the epididymis via the luminal system and play an important role in maintaining the function of epithelial cells, particularly in the initial segment. Previous work showed that {gamma}-glutamyl transpeptidase (GGT) mRNA IV, which is highly expressed in the rat initial segment, may be under the control of luminal fibroblast growth factor 2 (FGF-2) from the testis. The current studies were undertaken to identify which fibroblast growth factor receptors (FGFRs) are present in the principal cells of the rat initial segment and to identify other potential ligands for these receptors in rat rete testis fluid (RTF). Immunoblot analysis revealed that FGFRs 1–4 were present, and reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that both the IIIb and IIIc splice variants of FGFRs 1–3 were expressed. However, RT-PCR using RNA isolated from principal cells collected by laser capture microdissection revealed only FGFR-1 IIIc. Additional PCR analysis established that both the {alpha} and ß forms of FGFR-1 IIIc were expressed in principal cells. Both FGF-4 and FGF-8 were present in rat RTF, as determined by immunoblotting. Thus, FGF-2, -4, and -8, found in RTF, may act upon FGFR-1 IIIc in the principal cells of the initial segment to regulate GGT mRNA IV expression.

epididymis, growth factors, kinases, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interactions of growth factors with their specific cell surface receptors influence many cellular functions, including gene expression. Growth factors produced by the testis reach the epididymis via rete testis fluid (RTF) where they may interact with receptors on the apical surface of epididymal epithelial cells. These growth factors may be necessary for normal epididymal function particularly in the initial segment, which differs from the remainder of the epididymis in its dependence on luminal testicular factors. Efferent duct ligation (EDL), which prevents the entry of RTF into the epididymis, results in cellular changes in the initial segment with no effect on the remainder of the epididymis [1–3]. In addition, multiple genes expressed in the initial segment are under the control of luminal testicular factors.

In the rat, {gamma}-glutamyl transpeptidase (GGT) is a single-copy gene whose protein can be translated from five different mRNAs (I–V), each derived from a different promoter [4]. Previous studies have shown that only GGT mRNAs II, III, and IV are expressed in the rat epididymis [5]. Whereas GGT mRNAs II and III are expressed mostly in the caput and are under the control of testicular androgens, GGT mRNA IV is highly expressed in the initial segment and is under the control of luminal testicular factors [5, 6]. EDL resulted in 50% reduction of GGT mRNA IV by 12 h and 90% reduction after 24 h [6]. Subsequent studies showed a significant decrease in overall GGT protein and enzyme activity levels in initial segments after 3 days of EDL [7]. Culturing the 3-day EDL initial segments in medium containing fibroblast growth factor (FGF) 2 restored GGT protein and enzyme activity to sham control levels, whereas epidermal growth factor had no effect [7]. Both rat RTF and initial segment luminal fluid contained a 16-kDa protein recognized by an FGF-2 antibody [7]. A 43-kDa FGF-2-like protein was also found in initial segment homogenates but disappeared after a 3-day EDL [7]. FGF-2 is made in the rat testis [8] and is thought to play a role in the development of the epididymis [9]. The activity of GGT in Sertoli cells can be regulated in part by FGF-2 [10]. In addition to FGF-2, several other FGFs are synthesized in the adult testis, including FGFs 1, 3–5, 8, and 14 [11–13].

The FGF family and its receptors (FGFRs) constitute a complex system that controls many physiological processes, including cell growth, migration, and differentiation. To date, there are >20 members of the FGF family, with four known FGFRs (1–4). The FGFRs have a large extracellular domain, a transmembrane domain, and a split tyrosine kinase domain (Fig. 1). The extracellular domain contains three Ig-like domains, I, II, and III, an acidic box, a CAM homology domain, and a heparin-binding domain. Increasing the complexity of the FGFR system are the multiple alternative splicing events that occur within the extracellular and cytoplasmic domains, several of which influence ligand binding and receptor function.



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  		FIG. 1. Schematic representation of an FGFR. Shaded ovals represent the three Ig-like domains (I–III). The hatched part of Ig III represents the region, which can be derived by one of two exons that encode for the IIIb or IIIc form, respectively. The open rectangles represent the acidic, the cell adhesion molecule homology, and the heparin-binding domains. TM is the transmembrane domain. The split tyrosine kinase domain is represented by the solid rectangles (K1 and K2). Positions of primers used in PCR amplifications are shown above. F1 and R1 were used to distinguish between the IIIb and IIIc forms of FGFRs 1–3. F2 and R2 were used to amplify FGFR-4. F3 and R3 were used to distinguish between the short and long forms of FGFR-1. The arrow designates the location of the VT site

The second half of the third Ig-like domain in FGFRs 1–3 can be derived from one of two exons, giving rise to the IIIb and the IIIc forms, respectively, which differ in their ligand-binding profile [14–18]. This splicing event does not occur within FGFR-4 [19]. The original FGFR-1 cloned from chicken contained three Ig-like domains in the extracellular domain, referred to hereinafter as the {alpha} form [20]. A shorter (ß) form, in which the exon encoding the first Ig-like domain was spliced out, was subsequently cloned [21–23]. Alternative use of a 5' splice donor site within the juxtamembrane domain results in an additional 6 base pairs (bp) that code for a valine and a threonine, designated the VT site [24–26], which plays a role in modulating downstream signaling events [25–28]. The purpose of this study was to determine which FGFRs are present within the rat epididymal initial segment epithelium and to characterize further the FGFs present in RTF, a potential source of luminal factors regulating epididymal function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Normal adult male Sprague-Dawley rats (Hilltop Laboratories, Philadelphia, PA) between the ages of 50 and 100 days were maintained in the University of Virginia vivarium on a 12L:12D cycle with free access to food and water. All experiments complied with the regulations set forth by the Animal Welfare Act (Public Law 91-579), the Guide for the Care and Use of Laboratory Animals (NRC, 1996) published by the Department of Health and Human Services, and the policies and procedures of the University of Virginia Institutional Animal Care and Use Committee.

Materials

Pefabloc was from Roche Applied Science (Indianapolis, IN). Benzamidine was from ICN Biomedicals (Aurora, OH). Primers were synthesized by Invitrogen (Carlsbad, CA). All other reagents were from Sigma (St. Louis, MO) unless otherwise specified.

Tissues

Rats were killed with CO2 gas. The proximal half of each epididymal initial segment containing zone 1a and most of zone 1b (for characterization of zones, see [29]) was removed and immediately frozen in liquid nitrogen. Tissues were stored at -80°C until use.

FGFR and FGF Immunoblotting

For FGFR characterization, epididymal initial segments were ground with prechilled mortars and pestles under liquid nitrogen and lysed with RIPA buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, 0.1% SDS, and 1 mM EDTA) containing the following proteinase inhibitors: 1 mg/ml pefabloc, 1 µg/ml leupeptin, 10 µM pepstatin, 2 µg/ml aprotinin, and 10 mM benzamidine. The samples were incubated on ice for 15 min and then cleared by centrifugation at 16 100 x g at 4°C for 15 min. Protein determinations of the supernatants were made using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Forty micrograms of protein in Laemmli sample buffer (60 mM Tris HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 5% ß-mercaptoethanol) was loaded onto 6% SDS-polyacrylamide gels and transferred onto 0.45-µm nitrocellulose membranes (Bio-Rad) in 25 mM Tris base, 192 mM glycine, and 20% methanol at 4°C for 1 h at 100 V. Prior to blotting, the membranes were stained with Ponceau S to insure equal protein loading. Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline (TBS) plus Tween (TBST: 10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20). The blots were incubated with primary antibody overnight at 4°C in 5% nonfat dried milk in TBST. The following FGFR antibodies were used: 0.2 µg/ml FGFR-1 (Flg; H-76), 0.2 µg/ml FGFR-2 (Bek; C-17), 0.4 µg/ml FGFR-3 (H-100), and 0.4 µg/ml FGFR-4 (H-121). All FGFR antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The blots were incubated with 0.1 µg/ml peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) in 5% milk in TBST for 1 h at room temperature, rinsed in TBS, and then reacted with LumiGlo enhanced chemiluminescence reagent (Cell Signaling Technology, Beverly, MA).

For FGF characterization, RTF was concentrated by centrifugation through a 10-kDa molecular mass cut-off filter (Millipore, Bedford, MA) at 12 000 x g at 4°C for 10 min, which resulted in a 2.3-fold concentration of the RTF. Approximately 30 µg of protein in Laemmli sample buffer was loaded onto a 12% SDS-polyacrylamide gel. Immunoblotting was carried out as described above for the FGFRs. The following concentrations of antibodies were used: 5 µg/ml FGF-4 (AB-235-NA) from R&D Systems (Minneapolis, MN) and 1 µg/ml FGF-8 (N-19) from Santa Cruz Biotechnology. The FGF-8 antibody was preincubated with 5-fold excess of FGF-8 blocking peptide (Santa Cruz Biotechnology) in TBS for 1 h at room temperature prior to addition to the blot. The secondary antibody, peroxidase-conjugated anti-goat antibody (Chemicon, Temecula, CA), was used at 0.2 µg/ml in 5% milk in TBST.

Laser Capture Microdissection of Epididymal Principal Cells

Rats were killed with CO2, and the proximal half of the epididymal initial segment was removed and placed in Tissue-Tek O.C.T. compound (Sakura, Torrance, CA). The tissues were then frozen in isopentane chilled in a methanol/dry-ice slurry. Serial 10-µm sections of the rat epididymis were cut longitudinally on a Jung Frigocut 2800E cryostat at -20°C (Leica, Deerfield, IL) and mounted onto Superfrost Plus glass slides (Fisher Scientific, Suwanee, CA) at room temperature. Sections were immediately fixed in 70% ethanol for 30 sec, washed with distilled water, rinsed in 95% ethanol, immersed in filtered Eosin-Y (Richard Allen, Kalamazoo, MI) for 10 sec, dehydrated in 100% ethanol, and consecutively washed for 5, 10, and 15 min in fresh xylene. Slides were air dried for 1 h and transferred to a desiccator at room temperature. An Arcturus PixCell II laser capture microdissection (LCM) system equipped with an Olympus microscope (Arcturus Engineering, Mountain View, CA) was then used to capture principal cells (tall columnar epithelium with basally located nuclei) from epididymal sections. A single LCM cap (Capture Transfer Film TF100; Arcturus) was used per tissue section, and optimal conditions for LCM included a laser power of 40 mW and duration of 1.5–2.5 msec and laser spot size of 7.5 µm for epithelia. Captured cells were then mixed with TRIzol lysis buffer (Invitrogen) in a single Eppendorf tube, microcentrifuged, and stored as a lysate at -80°C. RNA was extracted within 72 h. The entire cell capture process, from tissue sectioning to tissue lysis, was rapidly completed to limit RNA degradation. Separate populations of principal cells were captured from the initial segments of four rats.

Isolation of Total RNA

The epididymal initial segments of four rats were ground with prechilled mortars and pestles under liquid nitrogen and placed into TRIzol lysis buffer. Laser-captured cells in TRIzol lysis buffer were thawed on ice. Isolation of total RNA was performed according to the manufacturer's instructions. The RNA pellets were resuspended in 15 µl diethyl pyrocarbonate-treated water. RNA was stored at -80°C until use.

Reverse Transcription Polymerase Chain Reaction

One microgram of total RNA from whole epididymal initial segment was subjected to treatment with 1 U amplification grade DNase I (Invitrogen) in 20 mM Tris HCl (pH 8.4), 2 mM MgCl2, and 50 mM KCl for 15 min at room temperature. The samples were then incubated with 2.5 mM EDTA at 65°C for 10 min. Synthesis of first-strand cDNA from total RNA was performed using the SUPERSCRIPT First-Strand Synthesis System for reverse transcription polymerase chain reaction (RT-PCR) (Invitrogen) according to the manufacturer's instructions. For whole initial segment, 8 µl of DNase-treated RNA was incubated with 1 mM dNTP mix and 50 ng random hexamers for 5 min at 65°C and immediately chilled on ice for 1 min. For laser-captured samples, 1.5 µl of the total RNA sample was used. PCR amplification was performed on 1 µl of a 1:10 dilution of the RT product from the whole initial segment and 1 µl of RT product from the laser-captured samples. PCR amplification of the FGFRs, GGT mRNA IV, and {alpha}M subunit of Mac-1 were performed in 20-µl reactions containing 60 mM Tris-SO4 (pH 8.9), 18 mM ammonium sulfate, 1.5 mM MgSO4, 0.1 mM each dNTP, 1 U PLATINUM Taq High Fidelity (Invitrogen), and 0.5 µM each primer (see Table 1). Multiplex reactions were set up with primers and competimers to amplify the 18S ribosomal subunit as an internal control (Ambion, Austin, TX). The Classic 18S Internal Standards (489-bp product) were used for reactions on FGFR-1 IIIc, FGFR-2 IIIb/IIIc, and FGFR-4. The Classic II 18S Internal Standards (324-bp product) were used for reactions on FGFR-1 IIIb and FGFR-3 IIIb/IIIc. All reactions were performed in a GeneAmp PCR System 2400 (Perkin Elmer, Foster City, CA) using the following conditions: 30 cycles of 94°C for 30 sec, 54°C for 30 sec, and 68°C for 30 sec, followed by a 2-min elongation step at 68°C. For PCR on the FGFR-1 IIIb variant, the following conditions were used: 35 cycles of 94°C for 30 sec, 57°C for 30 sec, and 68°C for 30 sec, followed by a 2-min elongation step at 68°C. All RT-PCR products were subsequently cloned into pCR-Blunt II TOPO (Invitrogen) and confirmed by sequencing (Biomolecular Research Facility, University of Virginia, Charlottesville, VA).


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  		TABLE 1. Primer pairs used for PCR amplification

RTF Collection

EDL was performed approximately 1–2 mm from the tunica of the testis, and 6–8 h later fluid was collected from the rete testis by micropuncturing the efferent ducts with a 30-µm glass pipette as previously described [30]. The fluid was centrifuged at 13 400 x g at 4°C for 8 min to remove spermatozoa. The supernatant was transferred to a fresh tube and stored at -20°C until use.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunoblot Analysis for FGFRs 1–4 in the Epididymal Initial Segment

To identify the FGFRs present in the rat epididymal initial segment, immunoblot analysis was performed. Immunoblotting was performed on initial segments isolated from three different rats, with identical results. Representative immunoblots demonstrating that FGFRs 1–4 are expressed in the initial segment are shown in Figure 2. Although the predicted molecular mass is approximately 130 kDa for the FGFRs, differential glycosylation and alternative splicing results in receptors of various sizes [21, 22, 31], which may explain the various sizes of the FGFRs (approximately 145 kDa for FGFR-1, 200 kDa for FGFR-3, 148 and 95 kDa for FGFR-2, and 152 kDa for FGFR-4). Similar results were obtained using rat testis protein as a positive control (data not shown) because this tissue has been shown to express FGFRs 1–4 [32]. Identical control blots incubated with secondary antibody alone displayed no immunoreactive bands (data not shown).



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  		FIG. 2. Immunoblot analysis of FGFRs 1–4 in rat epididymal initial segment. Protein from RIPA-extracted initial segment tissue was electrophoresed on a reducing 6% SDS-polyacrylamide gel and transferred to nitrocellulose. The membranes were probed in order with antibodies against FGFR-1, FGFR-3, FGFR-2, and FGFR-4

LCM of Initial Segment Principal Cells

The antibodies used in the immunoblot experiments do not distinguish between the IIIb and IIIc splice variants of these receptors. In addition, the cell type in which the protein is found cannot be discerned by this method. Therefore, principal cells were isolated from zone 1a by LCM. Figure 3 shows a typical frozen section before (Fig. 3A) and after (Fig. 3B) the capture. As a positive control, RNA from the principal cells was subjected to RT-PCR for GGT mRNA IV, which is expressed by this cell type. As a negative control, the principal cell RNA was also assessed for the presence of the {alpha}M subunit of Mac-1. This gene is expressed by basal cells found within the epididymal epithelium [33]. As shown in Figure 3C, both initial segment tissue and principal cells expressed GGT mRNA IV, whereas only initial segment tissue expressed the {alpha}M subunit of Mac-1, confirming the accuracy of our captures. These reactions were performed on each of the four LCM samples with identical results.



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  		FIG. 3. LCM of principal cells from the rat initial segment. Micrographs depict typical frozen sections used to capture principal cells from zone 1a in the initial segment. Bar = 40 µm. A) Before the capture. B) After the capture. C) RT-PCR was performed on total RNA isolated from rat epididymal initial segment tissue (T) and from principal cells taken by LCM (C) to assess the presence of GGT mRNA IV (GGT) and the {alpha}M subunit of Mac-1

RT-PCR for FGFRs Within the Initial Segment and Principal Cell Total RNA

To determine which of the FGFR isoforms were expressed in the epithelium, RT-PCR was performed on RNA isolated from initial segment tissue and principal cells. PCRs were performed as multiplex reactions with the 18S rRNA subunit serving as an internal control to show the presence of cDNA in the samples; however, these reactions were also performed with only the FGFR primers, yielding the same results. The IIIb and IIIc splice variants of FGFRs 1–3 and FGFR-4 were expressed in initial segment tissue (Fig. 4); however, only FGFR-1 IIIc (Fig. 4B) was expressed in the principal cell population. These reactions were performed on each of the four LCM samples with identical results. In one principal cell sample, we detected extremely low levels of FGFR-3 IIIb and IIIc but only after 35 cycles of PCR (data not shown). This discrepancy may be due to low levels of contaminating cells, such as narrow cells, even though care was taken to avoid cells with apically located nuclei.



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  		FIG. 4. RT-PCR for FGFRs 1–4. RT-PCR was performed on total RNA from rat epididymal initial segment tissue (T) and from principal cells taken by LCM (C). Primers were designed to screen for both the IIIb and IIIc forms of FGFRs 1–3 and for FGFR-4 (Table 1). The 18S ribosomal subunit was amplified as an internal control. Size standards are found in the far right lane of each gel. A) FGFR-1 IIIb. B) FGFR-1 IIIc. C) FGFR-2 IIIb and IIIc. D) FGFR-3 IIIb and IIIc. E) FGFR-4

Further Characterization of FGFR-1 IIIc Found in Principal Cells

Subcloning and sequencing of the FGFR-1 IIIc PCR products from each principal cell population was performed to check for the presence of the VT site. Of 20 clones that were sequenced, 18 clones were VT positive and 2 clones were VT negative. Additional PCR analyses revealed that both the {alpha} and ß forms of FGFR-1 were present in whole initial segment RNA and in RNA from principal cells (Fig. 5). The {alpha} form appeared to be more abundant, but this finding was not confirmed quantitatively. This analysis was repeated on all four LCM samples, with identical results.



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  		FIG. 5. RT-PCR for FGFR-1 ({alpha} and ß forms). RT-PCR was performed to screen for both the {alpha} (557 bp) and ß (298 bp) forms of FGFR-1 IIIc in total RNA from rat epididymal initial segment tissue (T) and from principal cells taken by LCM (C)

FGF Immunoblot Analysis on Rat RTF

To determine whether FGFs that can bind to FGFR-1 IIIc were present in rat RTF, immunoblots were performed using RTF collected from three different rats. Analyses were performed on the separate RTF collections, yielding identical results (Fig. 6). An approximately 23-kDa band was observed in the FGF-4 blot. Three bands of approximately 24, 29, and 34 kDa were observed in the FGF-8 blot (asterisks in Fig. 6). Preincubation of the FGF-8 antibody with a blocking peptide resulted in elimination of all three FGF-8 reactive bands. A blocking peptide was unavailable for the FGF-4 antibody; however, incubation with secondary antibody alone resulted in no detectable immunoreactive bands.



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  		FIG. 6. Immunoblot analysis of FGFs in rat RTF. Rat RTF (30 µg) was electrophoresed on a reducing 12% SDS-polyacrylamide gel and transferred to nitrocellulose. Membranes were blotted in order with antibodies against FGF-4, FGF-8, FGF-8 preincubated with a blocking peptide, and secondary antibody only. Asterisks to the left of the FGF-8 blot denote the three FGF-8 immunoreactive bands


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In initial studies, we determined that GGT mRNA IV is highly expressed in the initial segment of the adult rat epididymis and is under the control of luminal testicular factors, possibly by FGF-2 [5–7]. Here, we established which FGFRs are present in the initial segment of the rat epididymis, in particular the principal cells. Experiments also showed that other FGFs in addition to FGF-2 are in the luminal fluid that reaches the initial segment.

Immunoblot analysis revealed that FGFRs 1–4 are present in rat epididymal initial segment tissue. However, the antibodies used in these studies are incapable of distinguishing between the IIIb and IIIc variants of FGFRs 1–3, so RT-PCR was used to screen for the different isoforms of each receptor within initial segment RNA. Both the IIIb and IIIc isoforms of FGFRs 1–3 and FGFR-4 were expressed. GGT mRNA IV is expressed in the principal cells of the initial segment (unpublished observation). Therefore, LCM was used to isolate principal cells from this region. RT-PCR using RNA isolated from these cells identified FGFR-1 IIIc as the only FGFR present. These results were in contrast to those from several studies, in which the IIIc form of FGFR-1 was found primarily in the mesenchyme and the IIIb form was found in the epithelia [15, 34–36]. However, those studies were limited to evaluating cell lines and expression during development. The results presented here indicate that the IIIc form of FGFR-1 can be found in an adult epithelial cell type.

Further PCR analysis of the RT product from the principal cells revealed the presence of both the {alpha} and ß forms of FGFR-1 IIIc. Although some early studies indicated that FGF-2 binding was equivalent in both the {alpha} and ß forms [21, 31], study of recombinant proteins revealed that the {alpha} and ß forms differ in their affinity for both FGF-2 and heparin [37, 38]. Utilizing antibodies against the extracellular domain, Xu et al. [36] suggested that the tertiary structure of the {alpha} form was distinct from that of the ß form. Targeted disruption of FGFR-1 in mice led to embryonic death during gastrulation [39, 40], whereas mice with selective disruption of the FGFR-1 {alpha} form died on Embryonic Days 9.5–12.5, much later than did the FGFR-1 null embryos [41]. Furthermore, the switch from FGFR-1{alpha} to FGFR-1ß has been associated with the malignant progression of astrocytomas [42]. These results suggest that the biological activity of the {alpha} form is different from that of the ß form.

The FGFR-1 IIIc PCR products were also screened for the presence of the VT site. Of 20 clones analyzed, 18 contained the 6-bp coding region for valine and threonine. The two VT-negative clones were derived from separate samples, suggesting that both forms exist within principal cells, with the VT-positive form being more common. The VT site was shown to be part of a sequence necessary for binding of the scaffolding protein FRS2 [27, 28]. Mutation of the valine in this sequence and VT-negative splice variants failed to bind FRS2 [27, 28]. Through FRS2, FGFR-1 can modulate the Ras/ERK pathway by either recruitment of Grb2 [43] or atypical protein kinase C isoforms [44] and can also activate the phosphatidylinositol 3-kinase pathway [45]. Paterno et al. [46] demonstrated that the ratio of VT-positive to VT-negative receptors played a role in mesoderm formation in Xenopus, suggesting that the VT-negative form may serve to modulate downstream responses. Whether the presence of the VT site affects downstream events in our system is uncertain. However, FGFR-1 IIIc might signal through the VT-positive receptor to the Ras/ERK pathway to control GGT mRNA IV expression. Phosphorylated ERK levels were decreased after EDL, indicating that the Ras/ERK pathway was also under control of luminal testicular factors [47].

Previous studies from our laboratory demonstrated the presence of FGF-2 in rat RTF [7]. FGF-2 restored GGT protein and enzymatic activity levels to sham control levels following EDL [7]. FGF-2 activates FGFR-1 IIIc in a manner equivalent to that of FGF-1, which is capable of activating all the FGFRs [18]. To identify other FGF candidates possibly involved in the expression of GGT mRNA IV, rat RTF was analyzed for the presence of FGF-4 and FGF-8. Although multiple FGFs can bind FGFR-1 IIIc, FGF-4 can activate FGFR-1 IIIc in a manner comparable to that of FGF-1 and FGF-2 [18]. FGF-4 was thought to be expressed only during development, but recent studies confirmed expression in adult mouse brain and testis [12] and in adult rat testis [11]. Immunoblotting for FGF-4 (Fig. 6) showed an immunoreactive band of approximately 23 kDa, consistent with the previously reported molecular mass of FGF-4.

FGF-8 was cloned as an androgen-responsive growth factor that can be alternatively spliced to yield several proteins of various sizes [48, 49]. The results presented here indicate that several isoforms of FGF-8 (proteins of 24, 29, and 34 kDa; Fig. 6) are synthesized by the testis and secreted into RTF. Both FGF-2 and FGF-8 control the expression of the transcription factor polyomavirus enhancer activator 3 (PEA3) and related family members ERM and ER81 in chicken [50], Xenopus [51], and zebrafish [52, 53]. Previous studies from our laboratory revealed that the promoter for GGT mRNA IV has multiple PEA3 binding sites involved in the expression of GGT mRNA IV [54]. PEA3 itself is under the control of luminal testicular factors [55], perhaps by FGF-2 or FGF-8 found in RTF.

Conflicting data exists about whether FGF-8 can bind to and activate FGFR-1. Mitogenic studies using the BaF3 cell line transfected with an FGFR-1 IIIc cDNA showed no response to any of the FGF-8 isoforms [18, 56, 57]. However, mitogenesis may not be the appropriate method for assessing FGF-8 responses. FGF-8 was unable to compete for binding with FGF-1 in competition studies carried out in the same cells, but these studies involved only the FGF-8b isoform [57]. Perhaps other FGF-8 isoforms can bind and activate FGFR-1 IIIc. A mutant FGFR-1 IIIc was activated by FGF-8 in a mouse mammary carcinoma cell line [58].

The present results confirmed that FGFRs are present in the initial segment of the rat epididymis. FGFR-1 IIIc was found in the principal cells of the epithelium, cells responsible for GGT mRNA IV expression. FGFs 2, 4, and 8, potential ligands for FGFR-1 IIIc, were found in the fluid that enters the initial segment [7], supporting the idea that growth factors made by the testis enter the epididymis, where they may play an important role in the maintenance of initial segment function. Future experiments are aimed at determining the location of FGFR-1 IIIc within the principal cells (apical versus basolateral) and blocking the action of FGFR-1 IIIc to examine its role in controlling GGT mRNA IV expression.


   ACKNOWLEDGMENTS
 
The authors acknowledge helpful insight from Drs. R.J. Lye and C. Rodriguez of the Hinton laboratory and Drs. D. Brautigan, R. Ogle, A. Sutherland, and T. Turner.


   FOOTNOTES
 
1 This work was supported by NICHD grant HD32979 (to B.T.H.) and by NICHD/NIH through the assistance of the U54 Specialized Cooperative Centers Program for Reproduction Research: Molecular Biology Core Facility located at the University of Virginia, Charlottesville (U54 HD28934) and Lasercapture Microdissection Core Facility located at the University of Maryland School of Medicine, Baltimore (U54 HD36207). J.L.K. is supported by a grant from the Medical Scientist Training Program (NIH grant 2T32 GM07267). Back

2 Correspondence: Barry T. Hinton, Department of Cell Biology, P.O. Box 800732, University of Virginia Health System, Charlottesville, VA 22908. FAX: 434 982 3912; bth7c{at}virginia.edu Back

Received: 12 September 2002.

First decision: 12 October 2002.

Accepted: 28 January 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nicander L, Osman DI, Pløen L, Bugge HP, Kvisgaard KN. Early effects of efferent ductule ligation on the proximal segment of the rat epididymis. Int J Androl 1983 6:91-102[Medline]
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