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Research Article |
Departments of Urology3
Cell Biology,4 University of Virginia, School of Medicine, Charlottesville, Virginia 22908
ABSTRACT
Previous evidence has shown that sperm maturation is the result of successive events that influence sperm cells as they move through different microenvironments from the caput to the cauda epididymis. The physiological basis for the creation and maintenance of specific microenvironments along the epididymis are poorly understood. Anatomically, the epididymis consists of segments or lobules of epididymal tubule separated by connective tissue septa (CTS). The fact that CTS restrict the diffusion of tracer substances between segments and that certain gene expression patterns are segment-specific suggest that segments may represent functional epididymal units. In this report, we have further investigated epididymal segmentation by focusing on the ability of CTS to limit the effect of biologically relevant molecules, in particular epidermal growth factor (EGF), basic fibroblast growth factor (FGF2), and vascular endothelial growth factor A (VEGFA), in Segments 1 and 2 of the rat epididymis. We have demonstrated that these growth factors activate mitogen-activated kinase (MAPK) in both segments studied and that growth factors injected into the interstitial space of these segments in vivo exhibited a stimulatory effect only in the segment into which they were injected, i.e., MAPK activation was not observed in the adjacent segment. This restricting influence of CTS was abrogated by treatment with collagenase. In addition, we demonstrate the expression of selected forms of these growth factors and their receptors in Segments 1 and 2, and identify potential downstream targets. These results suggest that CTS regulate the trophic influences of growth factors and potentially other paracrine molecules, thus creating functionally separate units within the epididymis.
epididymis, growth factors, kinases, male reproductive tract, signal transduction
INTRODUCTION
The epididymis plays a central role in sperm maturation and storage of sperm cells. It is believed that different parts of this organ play different biological roles in relation to these processes [1]. In fact, sperm maturation appears to be the result of successive events that take place as sperm cells travel from the proximal to distal epididymis [2]. The epididymis is anatomically divided into caput, corpus, and cauda regions, which are further subdivided into intraregional segments that consist of lobules of epididymal duct surrounded by connective tissue septa (CTS) [3]. A recent development relevant to how different parts of the epididymis play specific biological roles has been the realization that the anatomical, intraregional segments of the epididymis are likely to be functional units within the organ [4, 5]. Experiments using tracer molecules have shown that diffusion in the interstitium between segments is severely restricted by the CTS [4], and it has been shown that the regionalization of biologically relevant properties of the epididymal duct, such as protein and gene expression, often coincide exactly with the limits imposed by the CTS [4, 6–10]. These observations have led to the hypothesis that segmentation of the epididymal interstitium by the CTS provides a physiological basis for epithelial segmental function by creating tightly controlled interstitial microenvironments within different epididymal segments. Separate microenvironments bordered by CTS may have particular relevance to intersegmental cell-cell communication. In particular, CTS may limit the influence of interstitial signaling molecules, such as growth factors, to the segment in which they were secreted.
Growth factors are polypeptides that promote cell division and differentiation, usually in a paracrine or endocrine fashion [11]. Growth factors mediate their effects through stimulation of the mitogen-activated kinase (MAPK) pathway, in which receptors with tyrosine kinase activity stimulate the GTP-binding protein RAS, which leads to the successive activation of RAF-1, MAP kinase kinase (MAP2K), and MAPK [12]. MAPK is a highly conserved kinase, which upon stimulation by phosphorylation accumulates in the cell nucleus and promotes the phosphorylation of numerous proteins and downstream kinases, including the transcription factors ELK-1, FOS, and MYC, which mediate many biological effects [13].
Recently, investigators have explored the possible roles of growth factors and their receptors in the epididymis [14]. Particular emphasis has been placed on expression and localization of growth factors or their receptors in the epididymides of different species. These growth factors include epidermal growth factor (EGF) [15–17], basic fibroblast growth factor (FGF2) [18–20], vascular endothelial growth factor A (VEGFA) [21, 22], nerve growth factor [23], platelet-derived growth factor [24, 25], transforming growth factor-ß [26–28], insulin-like growth factor [29, 30], erythropoietin [31], and hepatocyte growth factor [32, 33].
In the present study, we have focused on EGF, FGF2, and VEGFA, as previous evidence has indicated that these factors or their receptors are present in either the epididymal interstitial cells or in the tubule epithelium. We present evidence that CTS have a role in restricting the influence of interstitial signaling molecules to the segment in which they are first secreted, which helps explain how epithelial gene expression can be sharply regulated at segmental boundaries. Furthermore, we show that all three growth factors, EGF, FGF2, and VEGFA, induce rapid and sustained activation of the MAPK isoforms, MAPK1 (42-kDa isoform) and MAPK3 (44-kDa isoform), when perifused in vivo around the tubules of Segments 1 and 2 of the rat epididymis. In all cases, MAPK activation remained confined to the segment in which the specific growth factor was first presented. In addition, we demonstrate that all three growth factors and selected receptors are expressed in both Segments 1 and 2 of the rat epididymis, and that potential target genes are differentially expressed in the two segments depending on which growth factor is infused. These results focus new attention on the role of growth factors and interstitial cell-epithelial cell communication in the epididymis and suggest that the CTS play a role in limiting the effect of at least some paracrine factors to the segment(s) in which they are secreted.
MATERIALS AND METHODS
This work was conducted in accordance with the Guiding Principals of the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
In vitro Incubation
Adult Sprague-Dawley rats (500–600 g; Taconic Farms, Germantown, NY) were kept in a 12L:12D cycle with access to food and water ad libitum. Animals were anesthetized with sodium pentobarbital solution (i.p., 50 mg/kg, Nembutal; Abbott Laboratories, North Chicago, IL) and the epididymis and testis were exposed through a scrotal incision. Epididymides were extirpated and placed in ice-cold PBS. Under a dissecting microscope, Segments 1 and 2 (Fig. 1A) were decapsulated and separated along the plane of the CTS separating them. Both segments were incubated (32°C; 5% CO2, 95% air) in Eppendorf tubes that contained 200 µl of a medium [Medium M-199 (Gibco/Invitrogen, Carlsbad, CA)] constituted for epididymal tissue by supplementation with 10 mM sodium pyruvate (Gibco), 3 mM L-carnitine (Sigma, St. Louis, MO), 1 mM essential amino acids (Gibco), 5 µg/ml apo-transferrin (Sigma), 2 µM 5
-dihydrotestosterone (Sigma), 2 µM testosterone (Sigma)], in the presence or absence of 10–6 M human EGF (Invitrogen 13247–051), recombinant FGF2 (Invitrogen 13256–029), or recombinant rat VEGFA (Sigma V3638). Tissues were collected after 0, 30, 60, and 90 min of incubation. After incubation, segments were isolated, rinsed, and homogenized in a glass homogenizer fitted with a motor-driven Teflon pestle in 1 ml of ice-cold lysis buffer [PBS that contained 0.1% Triton X-100, 2.5 mM EDTA, 4 mM Na orthovanadate, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 25 µg/ml leupeptin (Sigma), and 5 µg/ml aprotinin (Sigma)]. Identical incubations were carried out as a control in medium without growth factors. In some cases, lysis was carried out in the presence of lambda protein phosphatase (New England BioLabs, Ipswich, MA) after growth factor stimulation, to confirm that the antibody against active MAPK (see below) was detecting only the phosphorylated protein. In these experiments, the segments were halved and lysed in 200 µl of lambda protein phosphatase reaction medium (New England BioLabs) supplemented with protease inhibitors (see below) and either 400 U lambda phosphatase or 4 mM Na orthovanadate. Protein samples were then used for electrophoresis and Western blotting, as described below.
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In Vivo Microperifusion
Anesthetized animals were prepared and the testis and epididymis were approached as above. The testis and epididymis together were placed in a specially designed, temperature-regulated (32°C) receptacle and the epididymis was prepared for in vivo micropuncture as previously described [34].
Microperifusions (perifusion is infusion into the interstitial space surrounding the tubules) were carried out using sharpened glass micropipettes with 50-µm tip diameter. The pipettes were filled with approximately 20 µl of growth factor solution (10–6 M EGF, FGF2 or VEGFA, as above, in PBS plus 0.1% lissamine green as a tracking dye). The loaded pipette was attached to a micromanipulator and to an infusion pump (Model 341b; Sage Instruments, Cambridge, MA) via a PE50 cannula. Under a dissecting microscope, the tunica albuginea of either Segment 1 or Segment 2 of the rat epididymis (see Fig. 1B) was punctured with the micropipette. The pipette tip was left in the segment's interstitial space and 15 µl of the selected growth factor solution was infused for approximately 5 min. The pipette was left in place until the end of the experiment and the epididymis was covered with mineral oil to prevent dehydration and to maintain proper tissue temperature (Fig. 1B). Identical experiments using vehicle alone as the perifusion medium were carried out as controls. To test the effect of CTS disruption on the effects of the perifused growth factors, 0.1% collagenase type I (Gibco) was added to the VEGFA solution only and used for in vivo perifusion into Segment 1 and Segment 2 in different epididymides for 90 min as described above. Perifusions with medium that lacked VEGFA but contained 0.1% collagenase were included as negative controls.
After each in vivo perifusion, Segments 1 and 2 were collected separately and analyzed for MAPK phosphorylation by Western blotting, as described below.
To provide a separate indication as to whether molecules with molecular mass within the range of the growth factors used in these studies could cross the CTS boundary, in vivo microperifusions were performed as described above but using the 26-kDa blue triosephosphate isomerase (TI, Sigma TI T9400; 3.5 µg/µl in PBS). Ten-microliter aliquots of the TI solution were perifused into either Segment 1 or Segment 2 of an epididymis (Fig. 1B). The segment not perifused in one epididymis was perifused in the contralateral epididymis. Beginning immediately after completion of the perifusion and continuing for periods lasting up to three hours, the segments were examined under a microscope to assess color development in the segment adjacent to the perifused segment. The appearance of blue coloration in the adjacent segment would indicate TI passage through the CTS.
Western Blotting
The concentrations of proteins in the tissue samples from the in vivo microperifusion experiments and the in vitro incubation experiments were determined according to the method of Bradford [35] using a commercial dye solution (Bio-Rad, Hercules, CA). Tissue samples (50 µg protein/lane) were electrophoresed in 12% PAGE gels, transferred to 0.2 µm nitrocellulose membranes (Bio-Rad), and stained with Ponceau S to verify the transfer. The membranes were then blocked with 3% non-fat dry milk in PBS and probed with primary antibody according to standard protocols. Active MAPK was detected using a rabbit antibody kindly provided by Dr. Thomas Sturgill (Dept. of Pharmacology, University of Virginia). This antibody specifically recognizes double-phosphorylated MAPK (pThr183-Glu184-pTyr185) in both the MAPK1 and MAPK3 isoforms [36]. For the detection of total MAPK, i.e., phosphorylated and nonphosphorylated forms, a rabbit anti-MAPK antibody was used (K23, sc-94; Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were subsequently washed and incubated with peroxidase-labeled, goat anti-rabbit antibodies (American Qualex, San Clemente, CA). Detection was carried out using a commercial chemiluminescent substrate (Pierce, Rockford, IL).
Real-Time Reverse Transcriptase-Polymerase Chain Reaction (Real-Time RT-PCR)
RNA extraction and cDNA synthesis were carried out as previously described [37]. Briefly, RNA was extracted from pooled samples (four tissues each from two animals) of Segment 1 and Segment 2 using a commercial phenol/guanidine isothiocyanate solution (TRIzol; Invitrogen) according to the manufacturer's instructions, and treated with DNase I (Invitrogen) for 15 min at room temperature. Complementary DNA (cDNA) was obtained using the SuperScript First-Strand Synthesis System (Invitrogen) using random hexamers according to the manufacturer's instructions. RT-PCR was performed in a MiniOpticon Real Time RT-PCR System (Bio-Rad) in 50 µl of a SYBR Green-based RT-PCR medium (iQ SYBR Green Supermix; Bio-Rad). The levels of mRNA expression for each growth factor and growth factor receptor were normalized to the expression level of the 18S ribosomal RNA, which was amplified using a commercial primer kit (QuantumRNA, Classic II; Ambion, Austin, TX). Quantitative gene expression was obtained from threshold cycle differences and the efficiency of amplification as described by Pfaffl [38]. Samples in which reverse transcriptase was omitted as well as water samples were included as negative controls. The probed transcripts, which were of similar length and C-G ratio, are detailed in Tables 1 and 2.
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RESULTS
In Vitro Activation of Epididymal MAPK by Growth Factors
In vitro experiments were performed using three growth factors, EGF, FGF2, and VEGFA, to 1) determine whether Segments 1 and 2 of the rat epididymis respond to all three factors, 2) confirm the approximate concentration of growth factor to use in the in vivo experiments, and 3) determine the timing and duration of MAPK phosphorylation in response to growth factor stimulation. All three growth factors activated (i.e., phosphorylated) epididymal MAPK1 and MAPK3 in Segments 1 and 2 of the rat epididymis (Fig. 2A). MAPK phosphorylation was usually undetectable in unstimulated tissues (0 min) but increased dramatically upon exposure to any one of the three growth factors. Total MAPK (phosphorylated and nonphosphorylated) did not change during the time course of the experiment (Fig. 2A). In all cases, the stimulation by growth factors persisted for the entire 90 min of the experiment (Fig. 1). Qualifying experiments included incubation times between 0 min and 30 min and demonstrated that stimulation of phosphorylation started as early as 2 min after exposure to growth factor (Fig. 2B). MAPK activation in tissues incubated without growth factors or incubated with growth factors and then treated with lambda phosphatase was typically either nondetectable or faint (not shown).
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These observations indicate that all three growth factors stimulate MAPK phosphorylation in Segments 1 and 2 of the rat epididymis, that the anti-active MAPK antibody detects only the phosphorylated form of the protein, and that the lack of detection of phosphorylated MAPK in unstimulated tissues is due to low levels of the phosphorylated form. These findings are confirmed by the fact that total MAPK immunostaining was present in unstimulated tissues and did not increase in stimulated tissues (Fig. 2A).
Both the MAPK1 and MAPK3 isoforms, also referred to as p42 and p44 [36], were detected in both Segment 1 and Segment 2 of the rat epididymis (Fig. 2). The average ± SD molecular mass of these two isoforms was 44.5 ± 0.11 kDa and 43.8 ± 0.28 kDa for MAPK1 and MAPK3, respectively (n = 5 for each). No other bands were revealed by the anti-active MAPK antibody or by the anti-total MAPK antibody in either the nonstimulated or growth factor-stimulated epididymides.
Activation of Epididymal MAPK Following Growth Factor Microperifusions In Vivo
Microperifusion of Segments 1 and 2 with a solution that contained TI, a 26-kDa blue molecule, demonstrated that molecules of this size have little, if any, access to adjacent segments, as assessed visually (Fig. 1B). This was true for both Segments 1 and 2, and restriction of TI movement across the CTS continued for up to 3 h after dye infusion.
MAPK activation was observed only in the specific segment perifused with either of the three growth factors, EGF, FGF2 or VEGFA, and not in the adjacent, non-perifused segment (Fig. 3). MAPK activation persisted through the entire 90-min experimental period, but regardless of whether the segment perifused was Segment 1 or 2 the adjacent segment never showed detectable levels of MAPK phosphorylation (Fig. 3). Preliminary experiments at 30 min and 60 min after growth factor perifusion gave the same result, i.e., at no time after growth factor presentation was MAPK activation detected in the non-perifused segment. As seen in the in vitro experiments, MAPK phosphorylation was not detected in segments perifused with medium alone (not shown). Of note, the responses of Segment 1 and Segment 2 to perifusion with any one of the three growth factors were similar (Fig. 3).
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To test the hypothesis that CTS are responsible for retaining the response to growth factors within the perifused segments, segments were perifused with VEGFA in the presence of collagenase. Ninety minutes after perifusion of the VEGFA/collagenase solution into either Segment 1 or Segment 2, MAPK activation was observed not only in the perifused segment but also in the adjacent segment (Fig. 4). Perifusions that contained collagenase alone did not activate MAPK in either segment, thus confirming that collagenase per se is not an activator of MAPK.
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Expression of Selected Growth Factors and Growth Factors Receptor mRNA in Segments 1 and 2 as Assessed by Real Time RT-PCR
Real Time RT-PCR revealed the expression of EGF and selected isoforms of Fgf2 and Vegfa in both Segment 1 and Segment 2 of the rat epididymis, as well as selected receptor isoforms (Table 3). Egf and Egfr transcripts were detected in both Segment 1 and Segment 2, with Segment 2 having lower Egf expression than Segment 1 but higher expression of Egfr. The Fgf2 and Fgfr isoforms investigated all showed higher expression in Segment 2 than in Segment 1, with the level of Fgfr1 increasing most prominently between the two segments (Table 3). All three VEGF isoforms were detected, with Vegfa and Vegfb being more highly expressed in Segment 2 than in Segment 1. This was not the case with Vegfc. Vegfr1 and Vegfr2 were also expressed in both epididymal segments. Thus, the representatives of each growth factor family used in the in vivo MAPK activation studies are, in fact, expressed at the gene level in both segments of the epididymides studied. Electrophoresis of the PCR products for each gene studied revealed single products of the correct molecular mass. Controls that lacked test sample or that contained samples in which no reverse transcription reaction was performed showed no amplification products.
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Expression of Potential Target Genes After In Vivo Growth Factor Perifusions in Segments 1 and 2 as Assessed by Real-Time RT-PCR To identify potential downstream targets for growth factors in the epididymal segments, and to determine whether such targets respond differently to different growth factors in different segments, we used real-time RT-PCR to detect genes that are known to be growth factor targets in other tissues [44]. The gene expression levels of urokinase plasminogen activator (Plau), angiopoietin 2 (Angpt2), matrix metalloproteinase 2 (Mmp2), prostaglandin-endoperoxide synthase 2 (Ptgs2), tissue inhibitor of metalloproteinase 1 (Timp1), and interferon regulatory factor 1 (Irf1) were determined. Gene expression was measured in the segments 90 min after growth factor perifusion into either Segment 1 or Segment 2 as well as in the adjacent, not-perifused segment collected at the same time. The same segment without perifusion was collected from the contralateral epididymis. This allowed comparisons of stimulated and non-stimulated tissues from the same animal. Similar results from duplicate experiments demonstrated no detection of Angpt2, Ptgs2, Timp1, and Irf1 in control or stimulated epididymides using the stated probes and conditions. Alternatively, Plau and Mmp2 were expressed in both Segment 1 and Segment 2 of these same epididymides; the Plau expression level relative to that of 18S ribosomal RNA was approximately 3-fold higher in Segment 1 than in Segment 2, whereas Mmp2 expression was similar in both segments. Microperifusion of VEGFA or EGF stimulated far higher expression of both Plau and Mmp2 in Segment 1 than in Segment 2, while FGF2 stimulated higher Plau expression in Segment 2 (Table 4). As before, the gene expression values were calculated relative to those of the ribosomal 18S gene. The values listed are the mean gene expression values of the perifused segment relative to the expression value in that same segment in the contralateral, nonperifused segment of the same rat. The means reflect the results of duplicate experiments with each growth factor.
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DISCUSSION
Several reports have demonstrated that the patterns of gene and protein expression in the epididymis often coincide exactly with the CTS dividing the epididymis into intraregional segments [4, 6–10]. Although these segments have generally not received much attention, recent evidence suggests that intraregional segments not only define morphologically evident portions of the epididymal duct, but they also form separate compartments of the epididymal interstitium [4] and contain individual segments of the epididymis that can exhibit unique gene transcription profiles [5]. The present study was designed to test the hypothesis that the segmentation of the epididymal interstitium plays a role in the regulation of segmented tubule function.
These studies were conducted using molecules for which evidence is available to suggest that they or their receptors are present in the epididymis. As examples, a putative form of EGF [15] and the EGFR [16] have been reported in humans and in several primate species, respectively. FGFRs have been demonstrated in the rat caput epididymis [39], and FGF2-like activity is present in homogenates of bovine and human epididymides [19]. VEGFA is present in the human epididymis [21], and VEGFRs are present in both human and mouse epididymis [21, 22]. The present studies were performed in the rat because the size of rat epididymis allows the experiments to proceed with a minimum need to pool multiple samples for protein and RNA analysis.
The study was conducted using Segments 1 and 2 (Fig. 1A) of the epididymis because the CTS between these two segments separate clearly different epithelia with different responses to stimuli [40, 41]. The nomenclature "Segment 1 and Segment 2" is the result of a simple numbering of all 19 segments of the rat epididymis, which is similar to the numbering of the 10 segments of the mouse epididymis [5]. Rat Segments 1 and 2 correspond to zones 1A and 1B in the terminology of Reid and Cleland [40]. MAPK phosphorylation was used as a test of growth factor action because MAPK activation is the hallmark of growth factor signaling [12].
The experiments with TI demonstrated no detectable movement across the CTS of a dyed molecule of a size that lies within the range of growth factor sizes (Fig. 1B), and suggested the possibility that paracrine factors having similar molecular weights might also be retained in the segmental interstitium in which they are first secreted.
The in vitro experiments established that the selected growth factors are active in the epididymis and determined the time-frame within which the growth factors stimulate MAPK phosphorylation in that tissue. The important issue was to avoid missing the period of MAPK activation in the in vivo experiments, simply because activation had occurred either before or after the designated sampling time of the in vivo experiment. The in vitro studies demonstrated that MAPK activation was detectable as early as 2 min after growth factor perifusion and lasted during the entire 90 min of the experiment (Fig. 2).
Furthermore, the in vivo studies were needed to maximize the possibility of detecting MAPK stimulation in the segment adjacent to the perifused segment should significant growth factor diffusion occur across the intervening CTS. Two important issues were considered: the rate of diffusion of a particular growth factor across the CTS and the time-course of MAPK activation by that growth factor. Previous studies of molecular movement across the CTS [4] as well as the failure of TI to cross across the CTS in the present study (Fig. 1B) have indicated that the diffusion of growth factors across the CTS is likely to be slow, if it occurs at all. Moreover, our in vitro results show that growth factors activate MAPK quickly and in a sustained manner (Fig. 2). Thus, if a growth factor diffuses either quickly or slowly from the perifused segment, we would still anticipate detecting its MAPK activation in the adjacent segment at the 90 min collection time.
Epididymal Segments 1 and 2 both expressed the classical MAPK1 and MAPK3 isoforms and they were activated by growth factors both in vitro (Fig. 2) and in vivo (Fig. 3). This is the first report we are aware of that demonstrates a growth factor-MAPK signaling pathway in the epididymis, although this has been alluded to previously with regard to FGF signaling [20, 42]. The present report demonstrates, at least to the level of MAPK activation, that Segments 1 and 2 respond similarly to the three growth factors studied (Figs. 2 and 3). Interestingly, and consistent with our hypothesis that the CTS provide a functional barrier that confines paracrine effects to individual segments, the MAPK activation induced by EGF, FGF2, and VEGFA occurs only in the perifused segments in vivo, regardless of whether the perifusion is in Segment 1 or Segment 2 (Fig. 3).
CTS are formed by an intensely eosinophilic, fibrous material with few cells, a combination that has collagen as a major component. Accordingly, the perifusion experiments using VEGFA as the stimulating growth factor were repeated, but this time collagenase was added to the perifusion medium to degrade collagen, which reduced the resistance of the intersegmental barrier to molecule movement. Under these conditions, VEGFA activated MAPK in both the perifused segment and the adjacent segment, again irrespective of which segment was perifused (Fig. 4).
The fact that exposure of CTS to collagenase allowed MAPK activation activity in the segment adjacent to the perifused segment suggests an abatement of the intersegmental barrier and passage of VEGFA from the perifused segment into the adjacent segment. This type of movement never occurred with any growth factor in the absence of collagenase, thus reinforcing the idea that under normal conditions, the physical integrity of the CTS prevents substances that originate in one segment from diffusing freely into the next segment. The growth factors used in this study [EGF (6 kDa), FGF2 (26 kDa), and VEGFA (45 kDa)] have the same or higher molecular mass as molecules previously shown to be largely retained by intraregional segments [4]. Thus, the CTS are capable of restricting the movement of a variety of paracrine molecules that may be secreted within a particular segment.
To confirm the biological relevance of our observations, we investigated the expression levels of the growth factors used in this study and their receptors in Segments 1 and 2. In some cases, notably for FGF and FGFR, only selected forms were analyzed due to the large number of members that constitute the families of these molecules. Selection was based on preliminary evidence of either prominent expression in or differential expression between Segments 1 and 2 of the rat epididymis, as determined by microarray analysis of segmental gene expression in the rat epididymis, a study that is currently being completed in our laboratory (unpublished results). Real-time RT-PCR for 13 different growth factor and growth factor receptor genes demonstrated that numerous forms of the growth factors and growth factors receptors are expressed in both Segments 1 and 2 of the rat epididymis, with Segment 2 having more transcripts for each form of growth factor and receptor, with the exception of egf, which is more prominently expressed in Segment 1 (Table 3).
Among all the changes in growth factor and growth factor receptor gene expression reported here between Segments 1 and 2 of the rat epididymis, the greater than 11-fold increase in FGFR1 was the most prominent (Table 3). FGF-FGFR signaling is very complex, with each of four different FGFRs having a number of preferred ligands among the now more than 20 FGFs [43]. The ligation of different FGFs to different FGFRs can have different consequences, none of which are known in the epididymis. Interestingly, Kirby et al. [39] also found prominent expression of the FGFR1 gene, specifically the IIIc variant, in the region that is commonly referred to as "the initial segment" of the rat epididymis. This tissue, as commonly divided, actually consists of Segments 1–4 (Fig. 1a). Nevertheless, the results of the present study are consistent with those of Kirby et al. [39] in indicating a predominance of FGFR1 expression in the proximal segments of the rat epididymis. The degrees to which these gene expression levels translate to protein levels remain to be determined.
A survey of potential target genes for growth factors in the epididymis has demonstrated that Plau and Mmp2 are potential target genes in Segments 1 and 2 of the rat epididymis. Of the six potential target genes surveyed, no previous references were found to Angpt2 or Irf1 in the epididymis and they were not detected in the present study. Ptgs2 in mice [45] and TIMP2 protein in several domestic species [46] have not been detected in caput tissues, and the expression of these genes was not detected in the present study. PLAU protein in the mouse epididymis [47] and MMP2 protein in the caput of rams, stallions, and boars [46] have been previously reported, although their gene expression levels in these tissues have not been reported. The expression of both Plau and Mmp2 was detected in Segments 1 and 2 in the present study and both were stimulated by perifusion of growth factor into specific segments. Interestingly, the two target genes responded differently depending on the segment and the growth factor perifused (Table 4). These results show differential gene regulation and suggest that Mmp2 and Plau are potential targets for particular growth factors in selected segments of the rat epididymis. While protein remodeling in the luminal environment of sperm or even in the epididymal epithelial cells are possible activities of the MMP2 and PLAU proteins, their roles are at present unknown and remain of interest as topics for further investigation.
In summary, CTS limit the effect of interstitial paracrine molecules and provide a physiological rationale for the phenomenon of segmented epididymal gene expression. Furthermore, this report adds new information regarding the presence and activity of growth factors in the epididymis and their potential target genes. Further exploration of the growth factors, as well as their receptors, signaling pathways, and downstream targets in specific segments of the epididymis will be of interest given the role growth factors play in the biological activities of many tissues.
ACKNOWLEDGMENTS
We thank Dr. Jeffrey Lysiak for his critical reading of the manuscript, and Dr. Thomas Sturgill for the generous gift of the anti-active MAPK antibody.
FOOTNOTES
1 Supported by NIH grants P50-DK45179 and T32-HDO7382. 
2 Correspondence: Terry T. Turner, Department of Urology, University of Virginia School of Medicine, P.O. Box 800422, Charlottesville, VA 22908. FAX: 434-924-8311; ttt{at}virginia.edu
Received: 10 March 2006.
First decision: 18 April 2006.
Accepted: 12 July 2006.
REFERENCES
This article has been cited by other articles:
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  S. A. Jelinsky, T. T. Turner, H. J. Bang, J. N. Finger, M. K. Solarz, E. Wilson, E. L. Brown, G. S. Kopf, and D. S. Johnston The Rat Epididymal Transcriptome: Comparison of Segmental Gene Expression in the Rat and Mouse Epididymides Biol Reprod, April 1, 2007; 76(4): 561 - 570. [Abstract] [Full Text] [PDF]
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