Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print October 1, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.019232
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BIOLOGY OF REPRODUCTION 70, 168–177 (2004)
DOI: 10.1095/biolreprod.103.019232
© 2004 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Expression of Platelet-Derived Growth Factor (PDGF) in the Epididymis and Analysis of the Epididymal Development in PDGF-A, PDGF-B, and PDGF Receptor ß Deficient Mice

Sabrina Basciani, Stefania Mariani, Mario Arizzi, Marina Brama, Andrea Ricci, Christer Betsholtz, Cecilia Bondjers, Giulia Ricci, Angela Catizone, Michela Galdieri, Giovanni Spera, and Lucio Gnessi1

Department of Medical Pathophysiology,2 University of Rome "La Sapienza," Policlinico Umberto I, 00161 Rome, Italy Department of Medical Biochemistry,3 University of Göteborg, Medicinaregatan 9A, SE 405 30 Göteborg, Sweden Department of Experimental Medicine,4 Histology and Embryology Laboratory, II University of Naples, 5-80138 Naples, Italy Department of Histology and Medical Embryology,5 University of Rome "La Sapienza," 00161 Rome, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) {alpha}- and ß-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-ß loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-{alpha} immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-ß were absent. The cellular localization of PDGF-A within the epithelium and the {alpha}-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-{alpha} system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR {alpha}- and ß-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-{alpha} mRNAs were seen by reverse transcription–polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-ß were almost not detectable. During the first days of life, PDGF-B and PDGFR-ß genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-ß, and PDGFR-{alpha} were constant with the only exception of a progressive decrease of PDGFR-{alpha} in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-ß -/- mice were available, and no differences were seen in the epididymis of these animals, compared with wild-type littermates. Taken together, these data indicate that the PDGF system is highly expressed in the epididymis and suggest that PDGF could be involved in the maintenance of morphological structure and functional control of this organ.

developmental biology, epididymis, growth factors, kinases, male reproductive tract


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The platelet-derived growth factor (PDGF) family consists of four members, PDGF-A, -B, -C, and -D, which exert their action via binding to and dimerization of two receptor tyrosine kinases, PDGF {alpha}-receptors (PDGFR-{alpha}) and ß-receptors (PDGFR-ß) [1]. Biosynthesis and processing of PDGFs result in the formation of full-length disulfide-linked homodimers PDGF-AA, BB, CC, and DD and the heterodimer PDGF-AB. The PDGF-A and PDGF-C chains selectively bind PDGFR-{alpha}, whereas PDGF-D preferentially binds PDGFR-ß, and PDGF-B displays similar affinity for both receptors [2]. PDGFs are members of the PDGF/vascular endothelial growth factors (PDGF/VEGF) family of growth factors and are major determinants in connective tissue growth, survival, differentiation, and functional control [3, 4]. The PDGF genes are expressed by a variety of cell types in developing and adult vertebrates. In vitro, a selective list of PDGF actions on connective tissue cells includes migration, proliferation, contraction, and inhibition of gap junctional communication and alteration of cellular metabolic activities including matrix synthesis, cytokine production, and lipoprotein uptake [4].

Genetic analysis using gene-targeting approaches has provided important information on the normal physiological functions of PDGFs. Mice lacking PDGF-B and those deficient in PDGFR-ß develop similar phenotypes characterized by renal, hematological, and cardiovascular abnormalities and death at day 17–19 of embryonic development (E17–E19) [5–7]. The renal and cardiovascular defects are due, at least in part, to a lack of proper recruitment of mural cells (vascular smooth muscle cells, pericytes, or mesangial cells) to blood vessels [5–8]. About 50% of mice lacking PDGF-A exhibit a lethal phenotype before E10, whereas surviving animals show a complex postnatal phenotype including a lack of alveolar septation in the lungs [9], dermal and hair defects [10], reduced proliferation of oligodendrocyte progenitors, hypomyelination of the central nervous system [11], loss of adult-type Leydig cells, and spermatogenic arrest [12]. The phenotype of mice lacking PDGFR-{alpha} is more severe, with incomplete cephalic closure, impaired neural crest development, cardiovascular and skeletal defect, and edemas, leading to embryonic death around E8–E16 [13]. Interestingly, PDGFR-{alpha} has recently been implicated in testis cord organization and fetal Leydig cell development [14].

Concerning PDGF-C, although loss of function studies are still unavailable, the localization of the growth factor in certain epithelial structures and PDGFR-{alpha} in the adjacent mesenchyme implies a potential for paracrine signaling in the developing embryo. PDGF-C expression seems to be particularly abundant at sites of ongoing ductal morphogenesis, which may also indicate a function in remodeling of connective tissue at these sites [15]. The expression of PDGF-D partially overlaps with that of PDGF-C in the cortical area of the developing kidney [16]. The different receptor specificities of these two recently identified PDGFs may provide distinct signals for migration and proliferation of both interstitial cells progenitors expressing PDGFR-{alpha} and perivascular progenitor cells expressing PDGFR-ß. Thus, the data available suggest that PDGFs provide selective signals during embryonic development by acting on the PDGFR-carrying progenitor cells and allowing for their proliferation and spreading on PDGF-producing endothelial or epithelial sheet/tubes.

It has been suggested that this process involves proliferation and migration only, processes known to be regulated by PDGF in vitro, but it may involve other functions as well, such as cell survival or differentiation. Besides the data indicating the absolute requirement for mesangial cells formation in the knock-out (KO) PDGF-B/PDGFR-ß mouse and for Leydig cell recruitment in PDGF-A KO mouse, a number of studies have strengthened the importance of PDGF in different districts of the genitourinary apparatus. PDGFR-{alpha} expression is present in the collecting tubules within the medulla of the embryonic and adult kidney [17]. Staining for PDGF B-chain of the ureteric bud and at the superficial layers of the urothelium lining the renal pelvis and ureters [18] was seen. A widespread expression of PDGF A-chain by the epithelium of the ureter renal pelvis and collecting duct in the fetal human kidney, and by collecting ducts and urothelium lining the lower urinary tract in the adult human kidney [19] was also observed. Moreover, a preferential expression of PDGF-C mRNA in the metanephric mesenchyme of the murine embryonic kidney during epithelial conversion and PDGF-D in the fetal kidney have been reported [15, 16, 20]. In the testis, mRNAs for PDGF A- and B-chain and PDGFR {alpha}- and ß-subunit are expressed both prenatally and postnatally [21–23].

In vitro experiments in the rat have shown that PDGF enhances the testosterone production by Leydig cells [24, 25]; is chemotactic and mitotic for peritubular myoid cells [26, 27]; stimulates the extracellular matrix deposition [28]; and synergistically with transforming growth factor-ß stimulates contraction [29] by peritubular myoid cells; can be involved in spermatogenic cell differentiation [30] and gonocyte proliferation [31]; and participates in the seminiferous cord formation.

The common mesonephric origin of the epididymis and the kidney, together with the crucial role exerted by the PDGF in testicular development, prompted us to analyze the expression pattern of PDGF-A, PDGF-B, and PDGFR {alpha}- and ß-subunit genes in the developing rat and mouse epididymis, the immunohistochemical distribution of the corresponding proteins, and the phenotypic appearance of the epididymis in PDGF-A, PDGF-B, and PDGFR-ß KO mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Preparation

Sprague-Dawley rats and CD-1 mice were purchased from Charles River Italia (Calco, Italy). Animals were housed under controlled light (14L:10D) and temperature (21–22°C) with free access to food and water. Epididymis from animals of different embryonic (E) and postnatal (P) ages (E19.5, P5, P10, P22, P35, P60, and P90 day rats; E18.5, P5, P10, P22, P30, and P60 day mice) were fixed in Bouins solution, processed, and embedded in paraffin according to conventional techniques for immunohistochemistry or rapidly removed, weighed, and stored in liquid nitrogen until RNA preparation or protein extraction (protocol n. 04/2002). The postnatal organs were subdivided into three separate regions (caput, corpus, and cauda) before freezing. PDGF-A +/- [9], PDGF-B +/- [5], and PDGFR-ß +/- mice [6] were bred as 129Ola/C57 BL6, 129Ola/C57BL6J, and 129Sv/C57BL6J hybrids, respectively. Heterozygotes were intercrossed and offspring of different age and genotype were killed and fixed for histological analysis. Because of the different survival of the -/- animals, PDGF-B and PDGFR-ß KO mice were studied at E17.5 and PDGF-A KO from P10 to P42. The animal study protocols were approved by the institutional committees on animal care.

Immunohistochemistry

Immunohistochemistry was carried out on 5-µm-thick sections by the streptavidin-biotin immunoperoxidase method, using a commercial kit (Zymed Laboratories Inc., San Francisco, CA). The deparaffinized sections were incubated overnight in a moist chamber at 4°C with 1:100 dilution of the primary antibodies. The following antisera, reactive in mouse and rat, were used: affinity-purified polyclonal rabbit anti-PDGF-BB, -PDGFR-ß, -PDGFR-{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and -PDGF-AA (RDI, Research Diagnostics, Inc., Flanders, NJ) antibodies. Negative controls were processed in the absence of the primary antibody. Slides were developed using aminoethylcarbazole as chromogenic substrate, which is converted by peroxidase into a red to brownish-red precipitate at the sites of antigen localization in the tissue. The preparations were counterstained with hematoxylin, dehydrated, and mounted.

Western Blot Analysis

Frozen tissues were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 200 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin plus 1% Triton X-100), followed by centrifugation at 14 000 rpm at 4°C for 20 min. Supernatants were collected as whole-tissue lysates. Protein concentration was determined using the Bradford protein assay method. Equal amounts (100 µg) of sample proteins were subjected to 7.5% SDS-PAGE and electrotransferred to nitrocellulose membranes. The membranes were blocked with PBS containing 0.05% Tween-20 and 5% nonfat dry milk and incubated with the same primary antibodies used for immunohistochemistry in RIPA buffer for 1 h at room temperature. Anti-rabbit horseradish peroxidase-conjugate IgGs were used for detection by enhanced chemiluminescence, according to the manufacturer's instructions (Amersham Pharmacia Biotech, Buckinghamshire, UK). Secondary antibodies alone served as negative controls. Western analysis was performed on the samples from three epididymis from each species.

Reverse Transcription and Semiquantitative Polymerase Chain Reaction

Frozen tissues were homogenized and poly(A)+ mRNA was prepared by using a commercial kit (Micro-FastTrak, Invitrogen, San Diego, CA). One microgram of mRNA was reverse transcribed at 37°C for 1 h in a 25-µl reaction volume containing 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, 50 mM dithiothreitol, 0.5 mM deoxynucleotide triphosphates (dNTPs), 0.5 µg random hexamer oligonucleotide, 200 U Moloney murine leukemia virus reverse transcriptase, and 26 U ribonuclease inhibitor (Promega, Madison, WI).

ß-Actin was used as a constitutively expressed gene product for comparison of PDGF and PDGFR mRNA abundance between samples. Then 0.5 µl of reverse transcriptase (RT) products were amplified with 2.5 U of Taq DNA polymerase (Promega) and 20 µM specific ß-actin primers (Table 1) in 50 µl of reaction mix containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, and 10 mM each of dNTPs, as follows: 94°C, 1 min; 58°C, 1 min; and 72°C, 1 min. Reactions were temporarily halted at 25, 28, 30, 33, and 35 cycles, and 10 µl of polymerase chain reaction (PCR) products were removed from each tube. All products were then analyzed by 1.5% agarose gel electrophoresis. Quantitation of the signals was performed by densitometric analysis, using densitometry computer software (Kodak Digital Science 1D Image analysis software, Eastman Kodak Co., Rochester, NY). PCR signals revealed a strong linear relationship, and 30 cycles were chosen for further analysis. Dilutions of RT products were made where necessary, and the amplification procedure was repeated until all samples were standardized for ß-actin content. After standardization, PCR was performed using appropriately diluted RT products in 50 µl of the reaction mix by utilizing 20 µM of each rat or mouse PDGF and PDGFR primer (Table 1). Thermocycling conditions were: initial denaturation for 3 min at 94°C, 35 cycles of amplification because levels of PCR products increased in a linear fashion for up to 40 cycles for all the genes analyzed, 1 min denaturation at 94°C, different annealing temperatures for each pair of primers (Table 1), 1 min extension at 72°C, and a final elongation of 5 min at 72°C. Parallel RT-PCR reactions without reverse transcriptase were performed for each sample to confirm that PCR products resulted from cDNA, rather than from genomic DNA. All products were then analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining.


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  		TABLE 1. Summary of PCR primer sequences, positions, and annealing temperatures

Statistical Analysis

Comparisons between groups were evaluated by the Student t-test when appropriate. Data were expressed as mean ± SEM and were considered statistically significant if P values were 0.05 or less.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Localization and Immunoblotting of PDGF Ligands and Receptors in Rat and Mouse Epididymis

Figure 1 shows the immunohistochemical distribution of PDGF-A and -B chain and of PDGFR {alpha}- and ß-subunit in E19.5 rat (Fig. 1, A–D) and E18.5 mouse (Fig. 1, E–H) epididymis. PDGF-A immunoreactivity was localized in the epithelium (Fig. 1, A and E), whereas PDGFR-{alpha} was observed in the mesenchyme surrounding the epididymal ducts (Fig. 1, C and G). The faint PDGF-A staining in rat and mouse interstitial tissue surrounding the epididymal ducts was considered aspecific because the corresponding negative controls showed a similar background (Fig. 1, A and E, insets). No staining for either PDGF-B or PDGFR-ß was seen (Fig. 1, B, D, F, and H).



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  		FIG. 1. Immunohistochemical localization of PDGF-A, PDGF-B, PDGFR-{alpha}, and PDGFR-ß in fetal rat (A–D) and mouse (E–H) epididymis. In E19.5 rat and E18.5 mouse, epithelial cells show a diffuse cytoplasmic PDGF-A immunostaining (A, E), whereas PDGFR-{alpha} (C, G) is localized in the mesenchyme surrounding the epididymal duct. Arrows, Epithelial cells of the tubules; arrowheads, mesenchyme. Negative controls obtained by omitting the primary anti-PDGF-A antibody are shown in the insets of A and E. Note the lack of positive staining for either PDGF-B (B, F) or PDGFR-ß (D, H). Bars = 20 µm (A, B, D, F, H), 40 µm (G), 50 µm (C, E).

In the prepubertal (P22) rat, the ligand- and receptor-positive staining was uniformly distributed in the epithelium lining the ducts in all segments of epididymis, with a tendency to an apical disposition of the immunoreactivity (Fig. 2). The only exception was a more intense PDGF-A reaction in the narrow cells of the initial segment (Fig. 2A). The same staining distribution was found in organs from pubertal (P35) animals (data not shown). In the adult rat, this pattern of expression was confirmed unless PDGF-A and PDGFR-{alpha} did not react in the clear cells of the cauda and there was a lack of increased compartmentalization in the narrow cells of PDGF-A in the initial segment (Fig. 3). Moreover, the immunohistochemical staining of PDGFR-{alpha} was reduced in P60 animals (Fig. 3, C, G, and K) and almost disappeared in older animals (data not shown). In the area immediately surrounding the epididymal tubules, which is occupied by three to five layers of myoid cells, and in the mesenchymal cells between the tubules, the reaction was always negative.



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  		FIG. 2. Immunohistochemical localization of PDGF-A, PDGF-B, and PDGFR {alpha}- and ß-subunit in P22 rat epididymis. Light micrographs of the initial segment (A), caput (B–D), corpus (E–H), and cauda (I–L) epididymis immunostained with anti-PDGF-A (A, E, I) -PDGF-B (B, F, J), -PDGFR-{alpha} (C, G, K), and -PDGFR-ß antibodies (D, H, L). The epithelial principal cells of the tubules (black arrows) show an intense positivity in all the segments of epididymis for all the substances tested. A more intense PDGF-A staining in the narrow cells (white arrow) of the initial segment is seen (A). Arrowheads, Mesenchyme. Bars = 20 µm



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  		FIG. 3. Region-related immunoexpression of PDGF-A, PDGF-B, and PDGFR {alpha} and ß subunit in adult rat epididymis. Light micrographs of the initial segment (A), caput (B–D), corpus (E–H), and cauda (I–L) epididymis immunostained with anti-PDGF-A (A, E, I), -PDGF-B (B, F, J), -PDGFR-{alpha} (C, G, K), and -PDGFR-ß antibodies (D, H, L). A high immunoexpression of PDGF-A in the entire cytoplasm of the principal cells in the initial segment (A), corpus (E), and cauda (I) epididymis that is absent in the clear cells of the cauda (I) is seen. PDGFR-{alpha} shows a positive staining in the epithelium lining the ducts in all the segments of epididymis with the exception of the clear cells of the cauda (K). The intensity of the PDGFR-{alpha} reaction is clearly reduced, compared with the other stainings. A distinct immunoperoxidase reaction for PDGF-B (B, F, J) and PDGFR-ß (D, H, L) is observed over the principal cells along the entire organ. Arrowhead, Epithelial principal cells; white arrow, narrow cells; C, clear cells; M, myoid cells; Lu, lumen; IT, interstitium; S, spermatozoa. Bars = 40 µm (C, G) 20 µm (A, B, D, E, F, H, I, J, K, L).

In the prepubertal and pubertal mouse, the distribution pattern was almost superimposable to that seen in the rat (data not shown). In the adult mouse, the immunohistochemical localization of ligands and receptors resembled what was observed in the rat with two exceptions: the clear cells of the mouse cauda epididymis were PDGF-A and PDGFR-{alpha} positive and the reduction of PDGFR-{alpha} staining was not observed (data not shown).

Western blot analysis on rat and mouse adult epididymis homogenates showed the expected molecular mass bands for PDGF-A, PDGF-B, PDGFR-{alpha}, and PDGFR-ß demonstrating the specificity of the antibodies used and confirming the PDGF system protein expression observed in the tissue (Fig. 4). In the rat, a less intense PDGFR-{alpha} immunoreactive band, relative to the other bands, was also observed. Negative controls, treated in the absence of the primary antibody, were constantly negative (not shown).



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  		FIG. 4. Representative Western blot analysis of PDGF-A, PDGF-B, PDGFR-{alpha}, and PDGFR-ß from total protein isolated from P60 mouse and rat epididymis. One hundred micrograms of protein per lane were used. Positions of molecular weight markers expressed as kilodaltons are shown

RT-PCR of PDGF Ligands and Receptors mRNAs

To quantify changes in PDGF and PDGFR gene expression throughout epididymal development, variations in transcript levels were determined using a semiquantitative RT-PCR by normalization of the amount of PCR product for PDGFs and PDGFRs against that for ß-actin. E18.5 mouse and E19.5 rat epididymis expressed PDGF-A and PDGFR-{alpha} mRNA and almost undetectable levels of PDGF-B and PDGFR-ß (Fig. 5). The mRNA extracted from P5–P90 rat epididymis contained transcripts for both the PDGFR {alpha}- and ß-subunit and PDGF A- and B-chain genes (Fig. 6A). The overall trend in PDGF-B and PDGFR-ß mRNA expression throughout postnatal development showed that, after an initial increase, the transcript levels were relatively constant without significant variations assignable to the portion of tissue analyzed caput, corpus, or cauda (Fig. 6B). On the contrary, PDGF-A and PDGFR-{alpha} had a variable pattern of expression, showing a significant decrease of PDGFR-{alpha} and an increase of PDGF-A transcript levels from P60 onward (Fig. 6B). P5–P60 mouse epididymis expressed mRNAs for PDGF-A and PDGFR-{alpha} at constant levels (Fig. 7). PDGF-B and PDGFR-ß genes were also observed at low levels in the perinatal period followed by a statistically significant increase at later times. Regardless of the source, the specimens gave bands of the expected size.



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  		FIG. 5. RT-PCR of PDGF-A, PDGF-B, PDGFR-{alpha}, and PDGFR-ß mRNA expression in prenatal rat (E19.5) and mouse (E18.5) epididymis. Histogram shows densitometric analysis of RT-PCRs expressed as a ratio between the gene being analyzed and the constitutive gene ß-actin. Each value represents the mean ± SEM of three different samples, each run in triplicate



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  		FIG. 6. RT-PCR of PDGF and PDGFR mRNA expression in postnatal rat epididymis. A) Representative RT-PCR showing PDGF-A, PDGF-B, PDGFR-{alpha}, and PDGFR-ß mRNA expression in 5-, 10-, 22-, 35-, 60-, and 90-day-old caput, corpus, and cauda epididymis and respective ß-actin mRNA levels. B) Graphs showing densitometric analysis of RT-PCRs expressed as a ratio between the gene being analyzed and the constitutive gene ß-actin. Each point represents the mean ± SEM of three different samples, each run in triplicate. Circle, Caput; square, corpus; triangle, cauda. * = P < 0.05



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  		FIG. 7. RT-PCR of PDGF and PDGFR mRNA expression in postnatal mouse epididymis. A) Representative RT-PCR showing PDGF-A, PDGF-B, PDGFR-{alpha}, and PDGFR-ß mRNA expression in 5-, 22-, and 60-day-old caput, corpus, and cauda epididymis and respective ß-actin mRNA levels. B) Graphs showing densitometric analysis of RT-PCRs expressed as a ratio between the gene being analyzed and the constitutive gene ß-actin. Each point represents the mean ± SEM of three different samples, each run in triplicate. Circle, Caput; square, corpus; triangle, cauda. * = P < 0.05

Effect of PDGF-A, PDGF-B, and PDGFR-ß Gene Targeting on Mouse Epididymal Development

Newborn PDGF-A -/- mice are outwardly normal but fail to thrive after birth. Most die within a few days, but some individuals survive for as long as 6 wk, eventually dying of pulmonary failure. Because of the lethal phenotype of the homozygous mutants, our analysis was restricted to male mice recovered between P10 and P42, killed before the first signs of respiratory failure were apparent. From P10 to P18, no alterations in the histology of the epididymis was observed (not shown). Starting from P25, the surviving animals showed a profound, progressive modification of the microscopic appearance of the epididymal structure, compared with wild-type (WT) littermates (Fig. 8, A–D). The diameter of the tubules an their lumen was reduced, and the epithelial cells showed a multilayered deposition disorderly arranged. The interstitial tissue was relatively more abundant than control and, because of the spermatogenic arrest in the seminiferous tubules, the epididymis was devoid of sperm cells. These morphological effects were of the same amplitude in all the epididymal regions. PDGF-B and PDGFR-ß KO animals do not survive to birth, and the organs were thus analyzed at E17.5. No differences were observed between -/- and WT littermates at this time (Fig. 8, E–H). Two to four KO and control littermates for each group were analyzed.



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  		FIG. 8. Histology of the epididymis of PDGF-A, PDGF-B, and PDGFR-ß KO and control mice. Hematoxylin/eosin-stained sections of PDGF-A, PDGF-B, and PDGFR-ß WT epididymis (A, C, E, and G), compared with mutant epididymis (B, D, F, and H) at P25 (A and B), P42 (C and D), and E17.5 (E, F, G, and H). A, B, C, and D show sections of the caput epididymis. Note the reduction of diameter and lumen of the tubules and the epithelial cells disorderly arranged in P25 and P42 PDGF-A KO mice (B, C). No differences are observed between PDGF-B and PDGFR-ß KO and WT littermates at E17.5. Arrows, Epithelial cells of the tubules. Bars = 20 µm (A, B, C, D) 40 µm (E, F, G, H)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Rat and mouse epididymis expresses PDGF-A and PDGFR-{alpha} genes and proteins before birth, whereas PDGF-B and PDGFR-ß are absent. The prenatal immunohistochemical localization of PDGF-A in the epithelial cells and PDGFR-{alpha} in the mesenchymal cells surrounding the tubules confirms previous in situ hybridization data [12]. Postnatally, a positive immunostaining for PDGF-A/PDGFR-{alpha} and PDGF-B/PDGFR-ß was found in both rat and mouse, and the corresponding genes were expressed accordingly. There was a profound difference in the immunohistochemical distribution of the staining, compared with the prenatal organ, and the cellular localization of the immunoreactivity in the animal species analyzed showed a different distribution pattern after puberty. After birth, PDGF-B and PDGFR-ß were constantly and homogeneously expressed in the epithelium of caput, corpus, and cauda of rat and mouse. The corresponding genes were first detected at P5. This result coupled with the absence of PDGF-B/PDGFR-ß immunostaining and mRNA expression before birth indicates that the PDGF-B/PDGFR-ß system starts to be expressed shortly after birth.

PDGF-A/PDGFR-{alpha} were also localized in the epithelial compartment, but their cellular localization in the adult animals was distinctive. In fact, in the rat, the clear cells of the cauda were not reactive either for the ligand or the receptor. In the adult epithelium, in line with the RT-PCR and Western blot expression data, the PDGFR-{alpha} staining showed a profound intensity reduction, whereas PDGF-A increased. In the adult mouse, we did not see the typical absence of PDGF-A and PDGFR-{alpha} staining in the clear cells or the profound PDGFR-{alpha} reduction of positivity detected in the rat both at protein and mRNA levels. Thus, the cellular localizations of PDGF and PDGFR in mouse and rat epididymis are similar but not identical.

These species differences and the different cellular distribution before and after birth have already been described for other tissues [21, 33], and although we do not know their functional significance, knowledge of the PDGF localization diversities should be useful in view of the potential development of animal models of epididymal injury. Previous immunohistochemical studies on the localization of other proteins in rat and mouse epididymis have shown contradictory results. For example, discrepancies in the immunolocalization of carbonic anhydrase II attributed to tissue preservation and immunocytochemical techniques have been reported [34, 35]. However, this seems not to be the case for the PDGF system because both ligand and receptor positivity is either present or absent in organs exposed to the same methodological procedures. The pattern of expression of the PDGF ligands and receptors along with their cellular distribution seem to indicate that they might be involved in the development and functional control of the organ.

The epithelial localization of PDGF-A and the mesenchymal localization of PDGFR-{alpha} during the fetal period of life in both rat and mouse epididymis suggest that, in line with what was observed in many other tubular structures [21, 33], a PDGF-A/PDGFR-{alpha} epithelial-mesenchymal paracrine interaction might be ideally suited to serve this recognized basic mechanism of development. However, for a candidate gene to be unequivocally shown to be involved in organ development, three conditions are necessary. First, the gene must be spatially expressed properly relative to the developing organ. Second, the gene has to be temporally expressed in a correct manner. Finally, when that gene is disrupted, normal organ development must not occur. The absence of PDGF-B and PDGFR-ß immunostaining in embryonic rat and mouse epididymis led to the prediction of a lack of phenotypic effect of loss of function of the corresponding genes. Accordingly, we did not see any alteration in the epididymis of PDGF-B and PDGFR-ß -/- fetal mice, although the early postnatal lethal effect of PDGF-B and PDGFR-ß gene targeting did not allow the analysis of the epididymal appearance of KO animals later during development.

Mutation of PDGF-A results in defects in lung, central nervous system, and development of other organs during fetal life [2]. We did not see any apparent epididymis malformation in PDGF-A KO embryonic animals. This could be explained either with a redundancy of the systems involved in the development of this organ or possibly with the vicarious effects exerted by PDGF-C, the alternative ligand for PDGFR-{alpha}. However, starting from P25, the lack of PDGF-A caused severe phenotypic alterations of the epididymis, which consisted mainly in a reduction of the lumen, a multilayered and deranged disposition of the epithelial cells, and disappearance of microvilli. There are at least two possible explanations for this effect: a direct influence of PDGF-A/PDGFR-{alpha} on the final maturation of the organ or an indirect effect mediated by the reduction in testosterone production that is a known phenomenon in PDGF-A -/- animals. Concerning the second hypothesis, the function, differentiation, and structure of the epididymis require the presence of androgens [36]. For example, in adult rat and goat, the castration induces a regression and disordered arrangement of the epithelium and a disappearance of the microvilli [37, 38], whereas the addition of androgens to the human adult epididymis in vitro is followed by an epithelial hypertrophy associated with a multiplication of microvilli [39]. Thus, there is a profound similarity between the phenotypic appearance of the epididymis of PDGF-A -/- mice and the morphological effect of testosterone deprivation on the organ. The involvement of testosterone decrease in the morphological effects on the epididymis of PDGF-A null mutation could be confirmed or refused by replacing testosterone in the KOs; however, the scarcity of -/- animals and the impossibility to predict their survival did not allow the test of the hypothesis. Thus, the existence of a PDGF-A/PDGFR-{alpha}-mediated effect on the epididymal structural maintenance and/or function not testosterone dependent can not be completely ruled out.

Another interesting point is that PDGF and PDGFR expression could be involved in the epididymal growth responses and interstitial fluid pressure regulation by angiotensin. An intrinsic angiotensin-generating system in the epididymal epithelium of mammals has been found [40–42]. Both the genes for angiotensin II (ATII) receptor type 1 (AT1), AT1a and AT1b, but not that for the receptor type 2 (AT2), are predominantly expressed in the epididymis of mature rat. In contrast, only AT1a and AT2 are highly expressed in the epididymis of immature rat. Consequently, type 1 subtypes may play a role in regulation of electrolyte and fluid transport in mature rat, whereas AT2 may be important in growth and development in the immature rat [41]. The notion that the ATII-mediated regulation of cell proliferation, cell growth, and fluid homeostasis is exerted through activation of PDGFs and PDGFRs [43, 44], and the recognition that the composition of the epididymal fluid is partly determined by the active secretion of anions, which is subject to control by a number of vasoactive peptides and ATII in particular [41, 45], suggests that the ATII-mediated effects on epididymis could be exerted through PDGF.

The mammalian epididymis provides a specialized microenvironment necessary for sperm maturation. Regional variation in the luminal proteins have been detected in several species, and these differences are the result of an active process of absorption and secretion by the epididymal epithelium controlled by androgens [36]. However, other factors such as proteins produced from specific parts of the epididymis may regulate the secretions in the various zones of the epididymis, leading to region-specific protein secretion [46]. For example, in the boar, procathepsin L, specific to the distal caput, disappears after castration and is not reinduced after testosterone treatment [47]. It has been postulated that PDGF can directly regulate its synthesis and secretion [47]. Thus, PDGF might be also involved in the regulation of protein secretion by the epithelium of the epididymis.

In conclusion, the discrete epididymal expression of PDGFs and PDGFRs in different developmental stages suggests that their regulation may have a role during the growth and development of the epididymis. The morphological changes seen after PDGF-A gene targeting demonstrate an influence, at least for this ligand, in the development and functional control of the organ. The intricate interactions among PDGFs and PDGFRs and other locally produced substances and their consequences on anion composition, fluid secretion, and development of the epididymis may provide the basis for the understanding of additional mechanisms regulating the locally restricted functions of the epididymis other than that exerted by androgens.


   FOOTNOTES
 
1 Correspondence: Lucio Gnessi, M.D., Ph.D., Dipartimento di Fisiopatologia Medica, Policlinico Umberto I, Università di Roma "La Sapienza" 00161, Rome, Italy. FAX: 39 06 4461450; lucio.gnessi{at}uniroma1.it Back

Received: 13 May 2003.

First decision: 11 June 2003.

Accepted: 10 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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