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BOR - Papers in Press, published online ahead of print October 15, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016816
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BIOLOGY OF REPRODUCTION 70, 340–347 (2004)
DOI: 10.1095/biolreprod.103.016816
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Hormonal Regulation and Functional Role of Vascular Endothelial Growth Factor A in the Rat Testis1

Stina Häggström Rudolfsson3, Pernilla Wikström4, Andreas Jonsson4, Ola Collin4, and Anders Bergh2,4

Department of Surgical and Perioperative Sciences,3 Urology and Andrology, Umeå University, Umeå, S-901 85, Sweden Department of Medical Biosciences,4 Anatomy, Clinical Chemistry and Pathology, Umeå University, Umeå, S-901 85, Sweden


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial cell growth factor (VEGF-A) is synthesized in the testis but its role and regulation in this organ have not been examined. VEGF and its receptors (VEGF-R) were quantified using reverse transcription-polymerase chain reaction and Western blot. VEGF, VEGF-R1, and VEGF-R2 mRNAs and VEGF protein were increased after treatment with 50 IU hCG. Injection of 100 ng human recombinant VEGF 165 into the testis caused an increase in endothelial cell proliferation, but only a moderate increase in testicular interstitial fluid volume. In contrast with systemic hCG treatment, local VEGF injection did not increase the permeability to intravenously injected colloidal carbon particles. However, if VEGF was given locally in the testes of animals pretreated with hCG 4 or 8 h earlier, VEGF acted in synergy with hCG to increase vascular carbon leakage by forming interendothelial cell gaps. Testicular blood flow was unaffected by local VEGF 165 injection. Treatment with a specific VEGF-R2 tyrosine kinase inhibitor blocked the hCG-induced increase in endothelial cell proliferation but did not affect the hCG-induced accumulation of polymorphonuclear leukocytes in testicular blood vessels or the increase in the testicular interstitial space. The present study demonstrated that testicular VEGF secretion is increased by hormonal stimulation of Leydig cells and that VEGF, through effects mediated via VEGF-R2, regulates endothelial cell proliferation in the rat testis. VEGF does not appear to regulate testicular blood flow and it is not involved in inducing the hCG-induced inflammation-like response in the testicular microvasculature. The permeability-increasing effect of VEGF is low in the testis under basal conditions but is apparently up-regulated by hCG treatment.

growth factors, human chorionic gonadotropin, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The testicular vasculature is in several ways unique. The resistance in the long and narrow testicular artery is unusually high, leaving capillary pressure in the testis lower than in other organs [1]. Because of a high vascular permeability, formation of interstitial fluid is still possible [for review, see 2, 3]. The mechanisms maintaining the high basal permeability are not fully established, but as the rat testicular endothelium is nonfenestrated and contains only few vesicles [2, 3], the permeability is apparently caused by the few open interendothelial cell junctions observed in capillaries and postcapillary venules [4, 5]. In addition to this, the testicular microvessels also show another remarkable feature: The endothelial cell proliferation rate is considerably higher than in other stationary organs [6]. The functional significance of this high proliferation rate and its regulation are largely unknown. Leydig cells, however, have a high capacity to stimulate angiogenesis. Leydig cells, in contrast with most other cells in the organism, induce a vigorous angiogenic response when transplanted under the kidney capsule [6, and references therein]. Leydig and possibly also other testicular cells secrete vascular endothelial cell growth factor (VEGF), and its receptors VEGF-R1 and VEGF-R2 are expressed on testicular blood vessels [6–10]. VEGF, also known as vascular permeability factor, is one of the most potent angiogenic and permeability-increasing factors known [11, 12]. The functional role and regulation of VEGF and its receptors in the testis, particularly in relation to high endothelial cell proliferation and vascular permeability in this organ, have not been studied. Indirect evidence does, however, suggest that VEGF could play an important role in testicular physiology and pathology. VEGF may be involved in mediating testicular growth and regression in seasonally breeding animals [13]. Transgenic mice overexpressing VEGF in germ cells show increased vascular density, spermatogenic arrest, and infertility [9, 14]. Long-term systemic or local VEGF treatment results in vascular growth [15] and edema [16, 17] in the testis. In the female gonad, VEGF synthesis is increased by LH/hCG treatment [18] and VEGF is essential for the vascularization of preovulatory follicles and the corpus luteum [19]. In the adrenal cortex, another steroid hormone-producing organ, the regulating hormone ACTH induces VEGF synthesis, and VEGF is responsible for maintaining endothelial cell fenestrations and a high vascular permeability [20]. The aim of this study was therefore to examine the functional role and regulation of VEGF in the rat testis. This was done by studying the hormonal regulation of testicular VEGF and VEGF receptors, by examining the effects of local injection of VEGF on the testicular vasculature, and by examining effects of blocking VEGF-R2 signaling.


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

Approximately 3-month-old male Sprague Dawley rats with a body weight of about 400 g were used (Taconic, Ry, Denmark). The rats were held in a controlled environment (12L:12D). Food and water were provided ad libitum. The animal experimentation ethics committee in Umeå approved the design of this study.

Experiment 1. Hormonal Regulation of VEGF and VEGF-Receptor Levels

Rats were randomly divided into five groups with five rats in each group. Each rat was injected subcutaneously with 50 IU hCG (Pregnyl, Organon, Västra Frölunda, Sweden), and 4, 12, or 24 h later, the rats were sedated with pentobarbital and the testicles were removed. The control group consisted of untreated rats. The testes were frozen in liquid nitrogen immediately after removal and stored at -80°C.

Quantitative Competitive Reverse Transcription-Polymerase Chain Reaction

Total RNA was prepared by the TRIzol method (Life Technologies, Täby, Sweden). Total RNA concentrations were measured using an ultraviolet (UV) spectrophotometer (Lambda 2 UV/VIS spectrophotometer; Perkin Elmer, Stockholm, Sweden). Truncated RNA of VEGF 165, VEGF-R1, and VEGF-R2 were used as internal standards (IS) and competitively amplified with VEGF, VEGF-R1, and VEGF-R2 in the reverse transcription-polymerase chain reaction (RT-PCR) as described earlier [21]. All IS were synthesized by the method of Häggström et al. and Celi et al. [21, 22]. Each RNA sample was titrated with three different amounts of IS (double samples), ranging between 0.01257 to 0.314 amol for VEGF, 0.019 to 0.477 amol for VEGF-R1, 0.014 to 0.352 amol for VEGF-R2. VEGF primers used in the PCR reaction were designed for simultaneous amplification of all isoforms, resulting in PCR products with lengths of 435 (VEGF 188), 363 (VEGF 164), and 231 (VEGF 120) base pairs (bp) [21]. PCR product lengths of R1 and R2 were 214 and 337 bp, respectively. Resulting PCR products were analyzed and mRNA levels were calculated from the linear regression by extrapolation at equivalent template to IS signals as previously described [21, 23].

Protein Extraction and Western Blot

Frozen testis tissues were homogenized in a microdismembrator (Mikro-dismembrator U; Biotech International, Braun, Sweden) and suspended in cold lysis buffer (0.5% NP-40, 0.5% NaDOC, 0.1% SDS, 50 mM Tris pH 7.5, 150 mM EDTA pH 8.0, 1 mM NaF) and complete protease inhibitor cocktail (Boeringer Mannheim AB, Bromma, Sweden). Supernatants were collected after 10 min refrigerated centrifugation at 20 000 x g, and stored at -20°C until used in the Western blot procedure. Total protein concentrations were determined with BCA Protein Assay kit (Pierce, Rockford, IL).

For Western blotting, protein samples were mixed with electrophoresis sample buffer containing 2% SDS and 5% 2-mercaptoethanol and boiled for 10 min. Forty micrograms of protein sample were electrophoresed on 12% SDS-polyacrylamide gel. Fractionated proteins were electroblotted onto a nitrocellulose membrane (Hybond-P; Amersham Pharmacia, Uppsala, Sweden) and the membrane was stained with 1% Ponceau-red in 0.05% acetic acid to ascertain equal loading of proteins. The membrane was blocked in 5% dry milk, 0.1% Tween-20 in PBS (PBS-T) for 1 h at room temperature prior to incubation with primary antibodies. VEGF was detected with a rabbit polyclonal antibody to VEGF (sc-152; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Actin was detected with a rabbit polyclonal antibody to actin (anti-actin, no. A2066; Sigma, Stockholm, Sweden). Primary and secondary incubations were carried out for 1 h at room temperature. Immunoreactions were detected using chemiluminescence, SuperSignal West Dura (Pierce) and quantified by densitometry using Fluor-S Multiimager and Quantity One software (Bio-Rad, Hercules, CA). Each sample was analyzed three times. Washing between steps was carried out using PBS-T. Molecular sizes of protein bands were determined by parallel electrophoresis of molecular weight markers (Bio-Rad Laboratories AB, Sundbyberg, Sweden).

Experiment 2. Effects of Intratesticular Injection of VEGF

Intact rats and rats that had been injected subcutaneously with 50 IU hCG 4 or 8 h earlier were sedated with pentobarbital (50 mg/kg). The testes were exposed via a scrotal incision. One hundred nanograms human recombinant (hr) VEGF 165 (R&D Systems, Abingdon, UK) dissolved in 50 µl sterile saline (0.9% NaCl) was injected in one testis and 50 µl saline was injected in the control testis. In situations where human VEGF was without effect, the effect of local injection of 100 ng murine recombinant (mr) VEGF 164 (R&D Systems, UK) was also examined. After intratesticular VEGF treatment, the following studies were performed.

Testicular blood flow Testicular microcirculation in both the VEGF- and saline-injected testis as well as in the contralateral testis was simultaneously studied using a two-channel laser Doppler flow meter (PF 4001 Master; Perimed, Stockholm, Sweden) connected to a personal computer as described earlier [24]. The average blood flow (measured in arbitrary perfusion units), -5 to 0 min before injection, was compared with that 0–20 min after injection by using the Perisoft for Windows software v. 1.01 (Perimed). In a similar way, the effects of intratesticular VEGF and saline were examined in rats that had been given 50 IU hCG 4 h earlier.

Testicular vascular permeability a) Interstitial fluid volume: Changes in the volume of testicular interstitial fluid are an index of changes in testicular vascular permeability [25]. The volume of testicular interstitial fluid was therefore measured 1 and 2 h after local injection of VEGF in one testis and sterile saline in the contralateral testis using methods previously described [25].

b) Labeling of open interendothelial cell junctions: In order to investigate the mechanism by which changes in vascular permeability occur, colloidal carbon (1 ml/kg body weight; Pelikan drawing ink; Hannover, Germany) was injected intravenously immediately after the intratesticular injections. One hour later, the animals were killed and the testes were removed and fixed in 4% formaldehyde, 3% glutaraldehyde, and 0.05% picric acid in 0.05 M cacodylate buffer [for details, see 4]. Colloidal carbon particles are too large to penetrate an intact endothelium but they penetrate and label open interendothelial cell junctions in the testis [4]. Previous studies have shown that local VEGF treatment in other tissues results in an increased vascular permeability that can be detected by this particular method [26]. The percentage of testicular microvessels labeled with carbon was measured using a light microscope at 1000x magnification as earlier described [27].

Testicular endothelial cell proliferation Immediately after local injection of VEGF in one testis and saline in the other, the animals were given an intraperitoneal injection with bromodeoxyuridine (BrdU, 50 mg/kg body weight; Sigma, St. Louis, MO). BrdU, a thymidine analogue, is incorporated into DNA in the S-phase of the cell cycle and the number of BrdU-labeled cells is an index of cell proliferation. Two hours later, the animals were killed. The testes were fixed in Bouin solution and embedded in paraffin. Five-micron-thick sections were immunostained with an antibody against BrdU (DAKO, Stockholm, Sweden) as earlier described [6]. The percentage of endothelial cells labeled with BrdU was calculated as described earlier [6].

Experiment 3. Effects of a VEGF-Receptor 2 Tyrosine Kinase Inhibitor (ZD6474) on hCG-Induced Changes in the Testicular Microvasculature

Adult Sprague Dawley rats were divided into four groups and treated as follows: 1) hCG 50 IU s.c. as described above. 2) hCG + ZD6474 (25 mg/kg s.c.). 3) ZD6474 (25 mg/kg s.c.). 4) Vehicle s.c. After 7 h, the animals were injected with BrdU (as described above). At 8 h, they were sedated with pentobarbital and the testicles were removed. The testes were weighed and fixed by immersion in Bouin for 24 h and embedded in paraffin. The volume densities of the interstitial space and polymorph nuclear (PMN) leukocytes in testicular blood vessels were determined by stereology as earlier described [28] and the percentage of BrdU-labeled endothelial cells as described above. Changes in the volume density of the interstitial space, which in the rat testis is dominated by large lymphatic spaces, can be used as an index of changes in vascular permeability [25]. ZD6474 is a selective VEGF-receptor 2 tyrosine kinase inhibitor developed by AstraZeneca. It inhibits VEGF-dependent angiogenesis in tumors and normal tissues [29]. The substance was kindly donated by Dr. Roger Henriksson, Gothenburg, AstraZeneca, Sweden.

Statistics

Statistical analysis was performed using the Mann-Whitney U-test for comparing groups and the Wilcoxon nonparametric test for comparing paired observations. Mean and standard error of the mean or standard deviations were calculated. A P-value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF and VEGF-Receptor Expression Following hCG Treatment

Quantification of VEGF, VEGF-R1, and VEGF-R2 mRNA Three isoforms of VEGF mRNA were amplified, VEGF 120, VEGF 164, and VEGF 188, in the PCR reaction. The lengths of the cDNA fragments were determined by using the TAMRA 2500 DNA size standard (Perkin Elmer) during the fragment analysis, and their identities were verified by sequence analysis (data not shown). The VEGF 188 isoform showed low expression and was not quantified, while VEGF 120 and VEGF 164 mRNAs were more abundant and expressed at approximately the same levels (Fig. 1A). There was an increase in the 120 and 164 VEGF isoforms at 4 and 12 h after hCG treatment compared with the control group (P < 0.05). VEGF-R1 and VEGF-R2 were up-regulated by hCG treatment and R2 showed significantly higher levels after 24 h of hCG treatment (Fig. 1B).



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  		FIG. 1. Competitive RT-PCR results showing VEGF (A), VEGF-R1 and VEGF-R2 (B) mRNA levels in 100 ng total RNA from control animals and hCG-treated animals. Values are presented as means ± SEM of five rats in each group. Both the VEGF and receptor levels were increased after hCG treatment. *, Significantly different from the control (P < 0.05) according to Mann-Whitney nonparametric test

Western blot VEGF and actin proteins were detected in the rat testis by using the Western blot technique (Fig. 2). The VEGF antibody recognized proteins with the approximate weight of 21 kDa in all groups. VEGF proteins showed higher levels after hCG treatment in the 12–24-h groups (Fig. 2).



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  		FIG. 2. Illustration of Western blot results showing VEGF and actin proteins in control testis tissue and after hCG treatment (A). Proteins with the weight of approximately 21 kDa were detected with the VEGF antibody. Relative VEGF protein levels in 40 µg total protein of control and hCG-treated rats, as quantified by densitometry (B). Values are presented as means ± SD of four to five rats in each group, and individual values were normalized for corresponding actin protein levels. VEGF protein levels were increased after hCG treatment. *, Significantly different from the control (P < 0.05) according to Mann-Whitney nonparametric test

Effects of Locally Injected VEGF 165 in the Rat Testis

Testicular blood flow Local injection of 100 ng hrVEGF 165 did not significantly influence average testicular blood flow 0–20 min after injection. Testicular blood flow in the VEGF 165-injected testis and the contralateral noninjected testis was 1.06 ± 0.06 and 1.02 ± 0.03 of the pretreatment values, respectively. Systemic blood pressure was 0.97 ± 0.07 (mean ± SD, n = 5 animals) of the pretreatment values. In the control animals, blood flow was 1.07 ± 0.08 in the saline-injected testis and 1.02 ± 0.003 in the contralateral control testis, while systemic blood pressure was 1.00 ± 0.06 (mean ± SD, n = 5) of the pretreatment values. VEGF or saline injection did not influence testicular vasomotion, i.e., the regular variations in microvascular flow. In order to exclude the possibility that the lack of effect of VEGF was related to the use of hrVEGF, we also injected the same dose of mrVEGF 164, but there was no significant effect on flow (data not shown). Human recombinant VEGF and saline were also injected into the testes of rats pretreated with hCG 4 h earlier. In the VEGF/hCG-injected testes, flow was 1.07 ± 0.40, and in saline/hCG-injected testes, flow was 1.04 ± 0.21 of the level seen before the intratesticular injection, demonstrating that local VEGF treatment did not influence testicular blood flow in hCG-treated animals.

Vascular permeability Testicular interstitial volumes measured 1 and 2 h after local injection of hrVEGF or control saline were 78.4 ± 8.3 vs. 67.5 ± 6.6 and 53.8 ± 7.7 vs. 56.0 ± 9.7 µl/g testis, respectively (mean ± SD, n = 5–7). The difference at 1 h between VEGF and saline treatment is statistically significant (P < 0.05), demonstrating that VEGF causes a transient, moderate (16%) increase in vascular permeability in the testis.

Intratesticular injection of 100 ng hrVEGF 165 (or murine recombinant, data not shown) in intact rats did not induce any apparent morphological signs of increased vascular permeability, at least not when examined by light microscopy. The number of blood vessels that were labeled with colloidal carbon was equally low in the VEGF-injected and the contralateral saline-injected testis (Fig. 3, Table 1), suggesting that VEGF alone is unable to open interendothelial cell gaps in testicular microvessels. There was also no sign of a VEGF-induced accumulation of leukocytes in testicular blood vessels. Similar results were observed after injection of mrVEGF (not shown). However, when hrVEGF was injected in the testes of animals that had received systemic hCG treatment 4 or 8 h earlier, the number of carbon leakage sites was higher in the VEGF than in the contralateral saline-injected testes (Table 1), demonstrating that VEGF acts in synergy with hCG to increase the number of open interendothelial cell gaps.



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  		FIG. 3. Toluidine blue-stained Epon embedded sections from testes of two rats injected locally with hrVEGF 165 in one testis (a, c) and saline (b, d) in the other 1 h earlier. Colloidal carbon was injected intravenously to demonstrate vascular leakage (x700 magnification). The first rat was an intact control (a, b), but in the second rat, 50 IU hCG was injected subcutaneously 4 h earlier (c, d). In the VEGF-injected testis in the intact rat (a), vascular morphology appears to be similar to that in the contralateral saline-treated testis (b) and no carbon leakage was observed. In contrast, carbon deposits (subendothelial deposits of dark foreign material marked by arrows) are observed in the saline-injected testes blood vessels in the hCG-treated animal (d), but the number and size of such leakage sites was further increased by local VEGF treatment (c)


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  		TABLE 1. Percentage of testicular microvessels with carbon leakage sites in hrVEGF- and saline-injected testes in intact animals and in animals treated with 50 IU hCG 4 or 8 h earlier

Endothelial cell proliferation Local injection of 100 ng hrVEGF 165 resulted in a significant increase (P < 0.05) in the percentage of BrdU-labeled endothelial cells 2 h after treatment compared with saline-injected contralateral testes, 7.7 ± 3.0 vs. 4.3 ± 1.9 (mean ± SD, n = 6 animals).

Effects of Inhibition of VEGF-Receptor 2 Tyrosine Kinase Signaling in the Rat Testis

Treatment with 50 IU hCG s.c. caused an expansion of the interstitial space (i.e., caused edema), an accumulation of PMN leukocytes in testicular blood vessels, and an increase in the endothelial cell BrdU-labeling index compared with that in vehicle-injected animals (Fig. 4, Table 2). Simultaneous treatment with ZD6474 blocked the hCG-induced increase in endothelial cell proliferation but left the other two responses unaffected (Table 2). Treatment with ZD6474 caused a slight decrease in endothelial BrdU-labeling index, but left the volume densities of interstitial space and PMN leukocytes unaffected compared with that in vehicle-treated animals (Table 2).



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  		FIG. 4. Sections from testes in an animal treated with hCG (a, c) or hCG + ZD6474 (b, d) 8 h earlier. Sections a and b (x275 magnification) are immunostained to localize BrdU. Human CG treatment is associated with an increase in the number of BrdU-labeled endothelial cells (v, vessel) but this response is blocked by treatment with ZD6474. Sections c, d (x140 magnification) are stained with hematoxylin and eosin. Human CG treatment is also associated with an accumulation of polymorphonuclear leukocytes in testicular blood vessels (arrows), but this response was not inhibited by blocking VEGF-R2 signaling with ZD6474. The interstitial space was of similar size in the hCG- and hCG + ZD6474-treated testes


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  		TABLE 2. Volume density (Vv) of interstitial space, volume density of PMN-leukocytes in testicular blood vessels, and endothelial cell BrdU labeling index in rat testes 8 h after treatment with hCG (50 IU s.c.), and hCG + ZD6474 (25 mg/kg s.c.), vehicle, or ZD6474 (25 mg/kg)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have shown that VEGF-A is synthesized in the testis [8], but its physiological role and regulation are largely unknown. We now demonstrate in the rat testis that hCG, a LH agonist that binds selectively to Leydig cells, causes an increased synthesis of VEGF mRNA and VEGF protein; within 12 h, VEGF may be synthesized by Leydig cells as this cell type expresses VEGF-mRNA [8], contains immunoreactive VEGF [6], and in contrast with other testicular cell types, induces angiogenesis when transplanted under the kidney capsule [6]. VEGF synthesis in other testicular cells (such as Sertoli cells, germ cells, or testicular macrophages) in response to increased Leydig cell activity is also a possibility because they have been shown to contain immunoreactive VEGF or VEGF-mRNA [6, 7, 9, 10]. Interestingly, hypoxia up-regulates VEGF synthesis in Sertoli cells [10] and LH/hCG treatment is known to cause a major decrease in blood flow 2–6 h after stimulation [see 2 for review], suggesting the possibility that increased VEGF synthesis could be secondary to tissue hypoxia.

We have previously demonstrated that hCG (or LH) treatment has several effects on the testicular vasculature. 1) The percentage of BrdU-labeled endothelial cells is increased [6]. 2) PMN-leukocytes accumulate in testicular postcapillary venules, plasma, and intravascular-injected colloidal carbon leaks; and PMN-leukocytes migrate through open interendothelial cell junctions in postcapillary venules, causing testicular edema and an inflammation-like reaction [2, 4]. 3) Blood flow is initially decreased, then increased [2]. Because hCG treatment increases testicular VEGF, it was of interest to study if some of the vascular effects of hCG could be mediated by VEGF.

Local injection of VEGF increases the number of proliferating testicular endothelial cells. It is therefore likely that the hCG-induced increase in testicular endothelial cell proliferation, first observed 8 h after treatment [6 and Bergh et al., unpublished observations], could be caused by increased VEGF. In line with this idea, transgenic overexpression of VEGF or local VEGF treatment results in an increased vascular density in the testis [9, 15]. VEGF is a potent endothelial cell mitogen in other tissues. This effect and apparently most other effects of VEGF are mediated by VEGF-R2 [30]. VEGF-R2 is present on testicular blood vessels [7] and the expression of this receptor was increased after hCG treatment. Blockage of VEGF-R2 signaling with a specific tyrosine kinase inhibitor inhibited the hCG-induced increase in endothelial cell proliferation, suggesting that VEGF is the principal mediator of this effect. Basal endothelial cell proliferation was, however, only moderately reduced by ZD6474 (and proliferation was still much higher than in other organs), suggesting that factors apart from VEGF are also responsible for maintaining the high basal endothelial cell proliferation in the testis. The nature of these factors remains unknown but several endothelial cell mitogens, e.g., endothelin-1 [31, 32] and the newly discovered endocrine-gland VEGF (EG-VEGF) [33] and its homologue Bv8 [34] are produced in the testis. Overexpression of EG-VEGF or Bv8 (not acting via VEGF-R2) results in a potent angiogenic response in the testis [34], suggesting that they could be involved.

Injection of VEGF caused only a slight transient increase in vascular permeability in the rat testis. The magnitude of this response is obviously lower than that in other rodent tissues where local injection of human VEGF 165 (in similar or lower doses than used in this study) causes a major increase in permeability and where VEGF is about 50 000-fold more potent than histamine [35]. In other tissues, VEGF increases permeability by inducing changes in the endothelium. Some of these can be marked with intravascular injected carbon and detected by light microscopy [26]. These changes, which occur rapidly after stimulation, include opening of interendothelial cell gaps [26] and formation of transcellular channels or fenestrations [36, 37]. In the testis, opening of interendothelial cell gaps after VEGF treatment was not observed. Formation of transendothelial cell channels or fenestrations, not visible by light microscopy, cannot be excluded. The endothelium in the rat testis is, however, in spite of basal VEGF secretion, nonfenestrated and contains few vesicles [2, 3]. There are no signs of fenestrations or increased number of endothelial cell vesicles in the testis after hCG treatment [2, 38] or in transgenic mice overexpressing VEGF in the testis [14]. Collectively, these observations suggest that some of the mechanisms by which VEGF influences the endothelium and increases permeability in other tissues are inhibited in the rat testis.

The testicular microvasculature is remarkably resistant to a variety of inflammation mediators that cause major changes in endothelial cell morphology and vascular permeability in other tissues [2, 3, 27]. We suggested that this difference could be caused by the presence of a permeability-inhibiting factor in testicular microvessels that is down-regulated by hCG [27, 39]. For example, local treatment with interleukin-1 does not cause vascular leakage under basal condition, but after hCG treatment, it markedly increases the number of blood vessels with open carbon-labeled interendothelial cell gaps [27]. Similarly, VEGF has a limited effect on its own, but after hCG treatment, it increases permeability by forming interendothelial cell gaps.

The hCG-induced inflammation-like response with accumulation of PMN leukocytes and edema, which starts already 4 h after treatment [2], was not prevented by blocking VEGF-R2 signaling. We have previously demonstrated that the hCG-induced increase in permeability is markedly reduced in PMN-depleted animals, suggesting that PMN leukocytes mediate the increase in vascular permeability [2]. The factor that causes leukocyte accumulation is unknown but is apparently secreted by Leydig cells [6]. In other tissues, VEGF is proinflammatory and causes leukocyte accumulation and adhesion to the endothelium [40, 41] and it could therefore be involved. Leukocyte accumulation was, however, maximal already at 4 h after hCG treatment [28] when VEGF protein levels were normal, indicating that other factors are probably more important than VEGF in attracting PMN leukocytes and inducing the testicular edema.

Intratesticular injection of VEGF did not cause any acute effect on testicular blood flow level or blood flow (vasomotion) pattern. This response is different from that in several other tissues where VEGF causes vasodilatation within minutes, principally by releasing nitric oxide (NO) [42, 43]. In contrast with the situation in other vascular beds, basal NO secretion is of limited importance in the regulation of testicular blood flow [44]. It is therefore possible that the lack of an acute dilatory response to VEGF could be due to a low acute NO-releasing capacity in the testicular endothelium under basal conditions [44].

In conclusion, this article demonstrates that testicular VEGF synthesis is increased after stimulation of Leydig cells and that VEGF, via effects mediated by VEGF-R2, could be involved (probably together with EG-VEGF/Bv8) in maintaining and increasing testicular endothelial cell proliferation, and, when necessary, promote vascular growth. VEGF may, to a limited extent, also be involved in maintaining and increasing vascular permeability, but most of the permeability-promoting and vasodilatory effects of VEGF observed in other vascular beds are absent in the testis under basal conditions. Some observations may explain this. Although most effects of VEGF in the vasculature are mediated by VEGF-R2 [30, 45, 46], the signaling pathways for the permeability and mitogenic effects are different [47, 48]. The permeability- (and flow-) increasing effect is mediated by increased NO [49–51], whereas the mitogenic response is mediated by another pathway. Low basal but increased endothelial NO secretion after hCG treatment [44] could consequently explain the permeability responses. Another factor that uncouples the permeability and mitogenic effect of VEGF in other tissues is angiopoietin-1 (ang-1) [16, 52], and we have shown that it may serve a similar role in the testis [53]. Ang-1 is expressed in testicular microvessels under basal conditions, local ang-1 treatment inhibits the hCG-induced increase in vascular permeability, and hCG treatment up-regulates the ang-1 antagonist angiopoietin 2 [53]. Interestingly, high basal levels of VEGF are present in several organs. In the salivary glands, which do not have a high vascular permeability, it is present apparently in the absence of active angiogenesis [54]. In the adrenal cortex, constitutive VEGF expression is associated with high vascular permeability and endothelial fenestrae but without endothelial cell proliferation [20]. One interpretation of such observations is that some of the vascular effects of VEGF could be moderated by other factors in tissues with constitutive VEGF secretion. The testis could be an example where the blood flow and permeability increasing (possibly mediated by NO and possibly counteracted by local angiopoietin 1), but not the mitogenic (mediated by other signaling pathways), effects of VEGF are more or less inhibited under basal conditions, but where the responsiveness to the permeability-increasing effect of VEGF can be increased by other factors up-regulated after hormonal stimulation of the Leydig cells.


   ACKNOWLEDGMENTS
 
Skillful technical assistance was given by Mrs. Sigrid Kilter, Mrs. Ulla-Stina Spetz, Mrs. Birgitta Ekblom, and Mrs. Elisabet Dahlberg.


   FOOTNOTES
 
1 This study was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Foundation, the Maud and Birger Gustavsson Foundation, the Swedish Society of Medicine, and the Medical Faculty, Umeå University. Back

2 Correspondence: Anders Bergh, Dept. of Medical Bioscience, Pathology, Bld. 6M, Second Floor, Umeå University, S-901 85 Umeå, Sweden. FAX: 46 90 785 2829; Anders.Bergh{at}medbio.umu.se Back

Received: 6 March 2003.

First decision: 25 March 2003.

Accepted: 23 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sweeney TE, Rozum JS, Desjardins C, Gore RW. Microvascular pressure distribution in the hamster testis. Am J Physiol 1991 260:H1581-H1589
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