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Testis |
Department of Surgical and Perioperative Sciences, Urology and Andrology,3
Department of Medical Biosciences, Pathology,4 Umeå University, Umeå, S-901 85, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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human chorionic gonadotropin, Leydig cells, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The functional role of continuous endothelial proliferation in a stationary organ such as the testis as well as its regulation remain unknown. Leydig cells, however, in contrast to most other cells in the body, induce a vigorous angiogenic response when transplanted under the kidney capsule [2 and references therein]. Leydig and other testicular cells secrete potent angiogenic factors, such as vascular endothelial cell growth factor-A (VEGF-A) [4–6], endocrine gland (EG)-VEGF [7], and endothelin (ET)-1 [8, 9]. The secretion of ET-1 increases after stimulation with hCG [8], but to our knowledge, the hormonal regulation of other angiogenic factors in the testis has not been studied. Local injection of a single dose of VEGF-A into the testis increases endothelial cell proliferation (unpublished results), but in contrast to most other tissues, in which VEGF-A cause a major acute increase in vascular permeability [10–14], permeability is only moderately increased in the testis (unpublished results). Moreover, testicular endothelial cells in rats do not show the morphological phenotype that generally is observed in VEGF-A-exposed endothelia, such as fenestrations and numerous transendothelial cell channels [1, 15]. These observations suggest that some as-yet-unidentified factor is able to inhibit the permeability-inducing, but not the proliferative, effect of VEGF-A in the testis.
The testis is an immune-privileged site. The immune-suppressed microenvironment is maintained by local secretion of immune regulators [16] and involves adaptations in the testicular microvasculature [1]. Testicular interstitial fluid induces an inflammatory response when injected subcutaneously, suggesting that some inflammation mediator is normally produced in the testis [17]. The effect of this mediator is, however, apparently neutralized in the testis under basal conditions, because no signs of ongoing inflammation are observed in normal, nonstimulated testes [17]. The testis is also remarkably resistant to the proinflammatory effects of several common inflammation mediators, such as histamine, leukotriene B4, serotonin, and interleukin-1 [18]. These observations suggest that the testis normally secrete an as-yet-unidentified inhibitor of inflammation-like vascular leakage [19].
Angiopoietin (ang) 1, which binds to the tie 2 receptor on endothelial cells, induces blood vessel maturation and stabilization during the later phases of angiogenesis [20]. Interestingly, ang 1 is able to block the permeability-inducing, but not the mitogenic, effect of VEGF [21]. In addition, ang 1 is able to block vascular leakage induced by inflammation mediators [21, 22]. Against this background, we wanted to test if ang 1 is expressed in the testis and whether this could explain why microvessels in this organ are resistant to the permeability-inducing effects of VEGF and common inflammation mediators. We demonstrate that testicular blood vessels express ang 1 protein under basal conditions. Angiopoietin 2 protein, which often acts as an antagonist to ang 1 [20], is also expressed in testicular microvessels at low levels under basal conditions, but its expression, together with that of VEGF-A, is up-regulated by hCG. In addition, intratesticular injection of human recombinant ang 1 is able to inhibit hCG-induced vascular leakage and edema in the testis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Approximately 3-mo-old male Sprague-Dawley rats (body wt, 
400 g) were used (Taconic M&B, Ry, Denmark). The rats were held in a controlled environment (photoperiod, 12L:12D). Food and water were provided ad libitum. Rats were injected subcutaneously with hCG (50 IU; Pregnyl; Organon, Västra Frölunda, Sweden) and examined 4, 8, 12, and 24 h later. In some rats, human recombinant ang 1 (933-AN; R&D Systems, Oxon, U.K.) or ang 2 (623-AN; R&D Systems) was dissolved in sterile PBS, and 1000 ng of human recombinant ang 1 or ang 2 (in 0.05 ml of PBS) were injected through the scrotal skin into the left testis and vehicle (0.05 ml of PBS) into the right testis in pentobarbital-anesthetized rats (see below). We injected 1000 ng because a dose of this magnitude is required to influence vascular permeability after local injection into the eye of diabetic rats, and the testis is considerably larger than the eye [23]. Some of these rats were simultaneously given 50 IU of hCG subcutaneously as described above and studied after 8 h. Previous studies have shown that treatment with hCG induced an inflammation-like vascular leakage in the testis [1, 24, 25]. To label leaking blood vessels, the ang- and hCG-treated animals were injected with colloidal carbon (1 ml/kg; Pelikan Drawing, Inc.) intravenously 1 h before the animals were killed, as described previously [24, 25]. The injected carbon stains the blood vessels with open endothelial cell junctions, but intact blood vessels are unstained [24]. Some animals were injected with bromodeoxyuridine (BrdU; 50 mg/kg; Sigma, St. Louis, MO) to label cells in the S-phase of the cell cycle, as described previously [2, 3]. The number of animals examined for each study is indicated in Tables 2–4. The local animal ethical committee in Umeå, Sweden, approved the design of the present study.
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Reverse Transcription-Polymerase Chain Reaction Quantification
Preparation of RNA standards To determine the copy number of the target transcripts of the samples, cloned plasmid cDNA for VEGF-A, ang 1, ang 2, and tie 2 were in vitro transcribed to RNA to generate an RNA standard curve. In brief, total RNA was reverse transcribed to cDNA in a 10-µl volume using AMV reverse transcriptase (Promega, Falkenberg, Sweden), 0.25 µg of random hexamers (Applied Biosystems, Stockholm, Sweden), and 10 U of RNasin (Promega) in the buffer supplied by the manufacturer. The reaction took place at 42°C for 60 min, followed by denaturation at 95°C for 7 min. Target template was amplified by conventional polymerase chain reaction (PCR) using Taq DNA polymerase (Roche Diagnostics, Bromma, Sweden) and specific primers (for sequences of all primers and PCR product lengths, see below and Table 1). The PCR conditions were 40 cycles of 95°C for 30 sec, 65°C for 30 sec, and 72°C for 45 sec, with the last cycle completed at 72°C for 10 min (PE 9600; Perkin-Elmer Applied Biosystems, Stockholm, Sweden). Resulting PCR products were cloned into pCRII vector using TOPO TA Cloning according to the manufacturer's instructions (Invitrogen, Stockholm, Sweden). Plasmids were purified using QIAprep Spin Miniprep Kit (Qiagen, Stockholm, Sweden) according to standard protocol and then sequenced (Big Dye Terminator Cycle on ABI Prism 377 Automated Sequencer; Perkin-Elmer Applied Biosystems, Stockholm, Sweden). Linearized plasmid was in vitro transcribed to RNA and stored at -80°C until use.
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Sample preparation Total RNA was prepared according to the TRIzol method (Invitrogen), and RNA concentrations were quantified spectrophotometrically at 260 nm (DU 640 Spectrophotometer; Beckman Coulter, Bromma, Sweden). The RNA integrity was studied by agarose gel electrophoreses and ethidium bromide staining.
Quantitative real-time reverse transcription-PCR Quantification of the mRNA coding for ang 1, ang 2, tie 2, and VEGF-A was performed using the LightCycler SYBR Green I technology (LC RNA amplification kit, catalog no. 2015137; Roche Diagnostics). Reverse transcription (RT)-PCR was performed in a 20-µl reaction volume containing 1x SYBR Green I buffer, 0.75x resolution solution, 0.4 µl of RT-PCR enzyme mix, 6 mM VEGF-A or 7 mM MgCl2 (reagents supplied by manufacturer), and 0.5 µM of each primer. The primers were designed by the nucleotide sequence relative to VEGF-A (GenBank accession no. M32977), ang 1 (GenBank accession no. AF030376), ang 2 (GenBank accession no. AF030378), and tie 2 (GenBank accession no. AF030423) (Table 1). The RT step was performed at 55°C for 10 or 30 min (ang 1), followed by an initial denaturation step at 95° C for 30 sec and then 45 cycles of 95°C denaturation for 0 sec, 65°C annealing for 10 sec, and 72°C extension for 13 sec. For ang 1, the fluorescence signal was acquired at a temperature 3°C below the melting temperature of the ang 1 peak to remove nonspecific primer-dimer product signal. Each experimental sample was run in duplicates. Negative controls were run concomitantly to confirm that the samples were not cross-contaminated. To confirm amplification specificity, the PCR products were subjected to a melting-curve analysis. The PCR products were quantified at the cycle number in which the fluorescent signals rose above background levels. The amount of target was determined from a corresponding RNA standard curve (see above), constructed with 10-fold serial dilutions (1 pg to 10 fg) in a background of 10 ng of MS2 RNA (catalog no. 165948; Roche Diagnostics). Data were analyzed using the LightCycler Software 3.3 (Roche Diagnostics).
Tissue Morphology and Immunohistochemistry
For immunohistochemistry, rats were sedated with mebumal (50 mg/kg) and fixed by vascular perfusion at a pressure of 1.2 m H2O with buffered formalin solution for 20 min. The testes were removed, weighed, postfixed for 24 h in the same fixative, cut in pieces, dehydrated, and embedded in paraffin. Sections (thickness, 4 µm) were immunostained using ang 1 (N18, sc-619; C19, sc-6320) and ang 2 (C19, sc-7015) goat antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) and a rabbit tie 2 antibody (A3124; CA) from Chemicon International, Inc. (Hofheim, Germany). After antigen-retrieval heating in a microwave oven for 20 min at 600 W in Target 1 solution (DAKO, Älvsjö, Sweden) for the ang antibodies and in citrate buffer (pH 6.0) for the tie 2 antibody, the sections were incubated overnight with the respective antibody (2 µg/mL). The immunoreactions were then visualized using LSAB kits from DAKO. The testes from three rats were examined for each antibody and treatment time point studied. Control sections were also immunostained with antibody that had previously been incubated with an excess of the corresponding peptides from Santa Cruz Biotechnology. The BrdU was immunolocalized, and the percentage of labeled endothelial cells was measured as described previously [2, 3].
For evaluation of the effect of intratesticular ang 1 or ang 2 treatment, animals were sedated with mebumal as described above. The testes were fixed by immersion in Bouin solution and embedded in paraffin (for stereological quantification of interstitial volume, which is an index of changes in vascular permeability [1, 26], and for accumulation of polymorphonuclear [PMN] leukocytes, as described previously [26]) or in 4% formaldehyde, 3% glutaraldehyde, and 0.05% picric acid in cacodylate buffer and then embedded in Epon (for visualization of carbon leakage, as described previously [24, 25]).
Statistics
The mean ± SD values were calculated. The SPSS 10.0 software (SPSS Sweden AB, Sundbyberg, Sweden) was used. Comparison among groups was done using the Kruskal-Wallis test. The Mann-Whitney U-test was applied for comparing groups (n = 4–6 animals per group). The Wilcoxon matched-pairs signed-rank test was used for paired observations. A P value of less than 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of ang 1, ang 2, tie 2, and VEGF-A mRNA was observed in the unstimulated rat testis. Their identities were verified by sequence analyses (results not shown). Treatment with hCG induced a marked increase in ang 2 and VEGF-A, whereas tie 2 and ang 1 mRNAs were largely unaffected (Table 2).
Localization and Regulation of Ang 1 and 2 and of the Tie 2 Receptor
Angiopoietin 1 A prominent ang 1 immunoreaction was observed at the luminal border of all types of intratesticular blood vessels—that is, in arteries, capillaries, postcapillary venules, and venules (Fig. 1). All other types of cells in the testis were unstained. The staining was apparently localized in the endothelial cells, but staining in pericytes cannot be excluded. After stimulation with hCG, the vascular ang 1 immunoreaction was largely unaffected, although a decrease in postcapillary venules was occasionally observed (Fig. 1). The two different ang 1 antibodies used gave the same result. Control sections, in which the ang 1 antibodies had been preincubated with ang 1 peptide, were unstained (not shown).
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Angiopoietin 2 In the normal, nonstimulated rat testis, an ang 2 immunoreaction was observed in the luminal border of all types of intratesticular blood vessels. The intensity was, however, greatest in arteries and low in capillaries and postcapillary venules. Stimulation with hCG caused an increased staining in the endothelial layer of all types of blood vessels that was evident at 8 and 24 h after treatment (Fig. 2), and because microvessels had low basal expression, this increase was most prominent in capillaries and postcapillary venules. Control sections, in which the ang 2 antibody had been preincubated with ang 2 peptide, were unstained (not shown).
Tie 2 In nonstimulated rat testes, a tie 2 immunoreaction was observed in the endothelial layer in all types of blood vessels (Fig. 3). The intensity and staining pattern of tie 2 was unaffected by hCG stimulation. Control sections incubated with unspecific rabbit IgG instead of tie 2 antibody were unstained (not shown).
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Effects of Intratesticular Ang 1 Treatment
Simultaneously with hCG treatment, 1000 ng of ang 1 were injected into the left testis and vehicle into the right testis, and effects were studied 8 h later. In the PBS vehicle-injected testis, numerous carbon-leakage sites were observed when the testes were examined under a dissection microscope [24] and in toluidine blue-stained Epon sections (thickness, 1 µm) (Fig. 4). The number of carbon-stained blood vessels was markedly reduced in the ang 1-injected testes (Fig. 4 and Table 3). In line with this, the volume density of PMN leukocytes accumulating in testicular blood vessels and the volume density of interstitial tissue (a marker of changes in testicular vascular permeability) [1], principally lymphatic spaces, was reduced in the ang 1 testes compared to the control testes (Fig. 5 and Table 3).
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Effects of Intratesticular Ang 2 Treatment
Local injection of 1000 ng of ang 2 in one testis and of PBS vehicle in the other resulted in a gradual increase in the volume density of the interstitial space, suggesting increased vascular permeability (Table 4). Carbon-leakage sites were, however, not observed in ang 2-injected testes, indicating that the reason for the increased interstitial space was not formation of interendothelial cell gaps. Injection of ang 2 increased the endothelial cell BrdU-labeling index, but it did not influence the volume density of PMN leukocytes in testicular blood vessels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular microvessels are remarkably resistant to the permeability-increasing effect of locally injected common inflammation mediators, such as serotonin, histamine, and interleukin-1 [17, 19], and also to VEGF-A (unpublished results). One of the characteristic effects of ang 1 in other tissues is that it stabilizes the endothelial cell layer and makes microvessels resistant to VEGF-A and inflammation mediator-induced increases in vascular permeability [21, 22]. Interestingly, high vascular ang 1 expression is apparently involved in maintaining the highly restrictive endothelial cell barrier in the central nervous system (i.e., in maintaining the blood-brain barrier) [28]. We therefore suggest that the ang 1/ang 2 balance, with ang 1 dominating in the testicular microvessels under basal conditions, could serve a similar role in the testis. Low local levels of ang 2 and higher levels of ang 1 and, possibly, of other inhibitors in testicular capillaries and postcapillary venules could possibly explain the resistance to permeability-increasing factors. It could also explain why testicular microvessels that are normally exposed to VEGF-A and to the newly discovered EG-VEGF [7] do not show the morphological phenotype (e.g., fenestrations and transendothelial cell channels) [1, 15, and unpublished results] observed in other vessels after VEGF exposure [see Introduction and 14].
Luteinizing hormone, or its agonist hCG, causes within 4 h a major increase in vascular permeability in the rat testis [1]. This increase is accompanied by accumulation of PMN leukocytes in testicular postcapillary venules, formation of large gaps between endothelial cells (that can be labeled with intravenously injected carbon) [24, 25], PMN migration into the interstitial space, and an interstitial edema [1, 24–26]. We now demonstrate that all these responses can be inhibited by ang 1. In PMN-depleted animals, the hCG-induced increase in vascular permeability is markedly inhibited, showing that the accumulating PMN leukocytes play an essential role in mediating the increase in permeability [29]. In the present study, we show that hCG treatment increases testicular VEGF-A expression. Interestingly, VEGF-A promotes adhesion of leukocytes to the endothelium, an effect inhibited by ang 1 [30], and accumulating PMN leukocytes may, in turn, secrete VEGF [31]. In the retina of diabetic animals, ang 1 treatment may, in addition to its endothelial-stabilizing effect, also inhibit leukocyte accumulation and vascular leakage by down-regulating VEGF synthesis [23]. In the testis, it therefore is likely that hCG-induced change in the microvascular ang balance may destabilize the endothelial cell layer and enhance the permeability-increasing effects of the accumulating PMN leukocytes and locally increased VEGF. A decreased ang 1:ang 2 ratio in testicular microvessels after hCG treatment may explain why hCG treatment turns resistant testicular microvessels into ones that respond to locally injected interleukin-1 [19] and VEGF-A (unpublished results). The present study indicates that local hormonal regulation of ang 2 and VEGF could be involved in the control of vascular permeability in the testis. In line with this, local injection of ang 2 increased the volume of the testicular interstitial space, suggesting an increase in vascular permeability [1, 26]. This increase in permeability was, however, not related to the formation of large interendothelial cell gaps and was of a lower magnitude than that seen after hCG treatment, suggesting that gap formation and hCG-induced vascular leakage are dependent on additional factors.
The proportion of proliferating endothelial cells in the testis is considerably higher than in other stationary organs [2, 3], and it is further increased 8 h after stimulation with hCG [2 and unpublished results]. The functional role and mechanism behind this is unknown, but Leydig and, possibly, other testicular cells apparently secrete large amounts of angiogenic factor(s) [2]. The nature of this factor is not fully established, but Leydig cells synthesize VEGF-A, EG-VEGF, and ET-1, which all stimulate angiogenesis, and the secretion of ET-1 [8] and VEGF-A (present study) is increased after stimulation of Leydig cells with hCG. In addition, ang 2 expression in microvessels is increased by hCG treatment, and ang 2 injection causes a moderate increase in endothelial cell proliferation in the testis. The functional significance of the high endothelial cell proliferation in the normal testis is largely unknown. Sprouting, bridging, or other morphological signs of new blood vessel formation do not, however, accompany the high endothelial cell proliferation, and cell proliferation is apparently balanced by endothelial cell apoptosis [2, 3]. This suggests the presence of some factor in the testis that normally inhibits new blood vessel formation in an angiogenic environment but that allows endothelial cell proliferation. Angiopoietin 1 may be involved in this. Under basal conditions, constitutively expressed ang 1 and low ang 2 may stabilize the microvascular endothelium [32], allowing proliferation (in response to locally produced endothelial mitogens, e.g., VEGF-A and EG-VEGF) but not other aspects of blood vessel formation. After hCG stimulation, a decreased ang 1:ang 2 ratio in testicular microvessels and increased VEGF-A and other factors may destabilize the microvascular endothelial cell layer, allowing gap formation, increased endothelial cell proliferation, and vascular growth. Vascular growth [33] and edema [22, 33] can also be induced in the testis after prolonged stimulation with locally or systemically delivered VEGF-A, indicating that the suggested ang 1 shield may be insufficient in this situation. The observation that ang 1 treatment inhibits vascular leakage in the testis suggests that it could, potentially, be used to prevent the testicular side effect of edema that is seen during systemic VEGF treatment for ischemic disorders [22, 34].
In summary, constitutive microvascular ang 1 expression, hormonally controlled levels of microvascular ang 2, and presence of other angiogenic factors like VEGF-A and EG-VEGF may be involved in maintaining the unique characteristics of the testicular microvasculature (high endothelial cell proliferation without angiogenesis and resistance to inflammation mediators) under basal conditions and to allow changes in this during gonadotroph stimulation. The regulation of the testicular microvasculature show some similarities to that in ovarian follicles, in which VEGF-A, ang 1, and ang 2 are also controlled by LH/hCG [35, 36] and in which these factors are involved in the growth and regression of the ovarian vasculature during the ovarian cycle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Anders Bergh, Dept of Medical Biosciences, Pathology, Umeå University, Umeå, S-901 85 Sweden. FAX: 46 90 7852829; anders.bergh{at}medbio.umu.se
Received: 15 November 2002.
First decision: 12 December 2002.
Accepted: 12 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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induces acute inflammation-like changes in the testicular microcirculation in adult rats. J Reprod Immunol 1990 17:155-165[CrossRef][Medline]This article has been cited by other articles:
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  H. Kanazawa, S. Nomura, and K. Asai Roles of Angiopoietin-1 and Angiopoietin-2 on Airway Microvascular Permeability in Asthmatic Patients Chest, April 1, 2007; 131(4): 1035 - 1041. [Abstract] [Full Text] [PDF]
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 H. Ishimoto, D. G. Ginzinger, and R. B. Jaffe Adrenocorticotropin Preferentially Up-Regulates Angiopoietin 2 in the Human Fetal Adrenal Gland: Implications for Coordinated Adrenal Organ Growth and Angiogenesis J. Clin. Endocrinol. Metab., May 1, 2006; 91(5): 1909 - 1915. [Abstract] [Full Text] [PDF]
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 A. Johansson, S. H. Rudolfsson, P. Wikstrom, and A. Bergh Altered Levels of Angiopoietin 1 and Tie 2 Are Associated with Androgen-Regulated Vascular Regression and Growth in the Ventral Prostate in Adult Mice and Rats Endocrinology, August 1, 2005; 146(8): 3463 - 3470. [Abstract] [Full Text] [PDF]
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M. Samson, F. V. Peale Jr., G. Frantz, N. Rioux-Leclercq, E. Rajpert-De Meyts, and N. Ferrara Human Endocrine Gland-Derived Vascular Endothelial Growth Factor: Expression Early in Development and in Leydig Cell Tumors Suggests Roles in Normal and Pathological Testis Angiogenesis J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4078 - 4088. [Abstract] [Full Text] [PDF]
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 S. H. Rudolfsson, P. Wikstrom, A. Jonsson, O. Collin, and A. Bergh Hormonal Regulation and Functional Role of Vascular Endothelial Growth Factor A in the Rat Testis Biol Reprod, February 1, 2004; 70(2): 340 - 347. [Abstract] [Full Text] [PDF]
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