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Involvement of vascular endothelial growth factor on spermatogenesis in testis with varicocele
Involvement of vascular endothelial growth factor on spermatogenesis in testis with varicocele The expression and the role of vascular endothelial growth factor in human testes with varicocele were examined. Vascular endothelial growth factor expression was increased in testes with varicocele, and was inversely correlated with total motile sperm count and testicular volume, indicating that excessive vascular endothelial growth factor has harmful effects on spermatogenesis in testes with varicocele. (Fertil Steril 2008;90: 1313–6. 2008 by American Society for Reproductive Medicine.)
Several mechanisms have been postulated to explain varicocele-induced deterioration of spermatogenesis. Hypoxia is known to be one of the factors involved in the pathophysiology of varicocele (1–3). Varicocele also alters testicular microcirculation to maintain adequate microenvironments in respond to hypoxia for spermatogenesis and steroidogenesis (4). However, few investigations have been done to examine the molecular events to address this issue. Vascular endothelial growth factor (VEGF) is a 21-kDa glycoprotein with a specific mitogenic effect for vascular endothelial cells (5), and is a major regulator of endothelial cell proliferation, angiogenesis, and vascular permeability (6). Vascular endothelial growth factor has a crucial role during testis morphogenesis through neurovascularization and cord formation (7). In human testis, VEGF has been shown to be expressed in the male genital tract including Leydig and Sertoli cells, certain epithelial and peritubular cells of the epididymis, and the epithelium of the prostate and seminal vesicle (8–10), and it exerts its biologic function by binding to tyrosine kinase receptor 1 (flt-1) and 2 (flk/KDR), which are located on the surface of endothelial cells, Leydig, and Sertoli cells (9). Increased expression of VEGF in testis has been reported in experimental varicocele using rats (2); however, the role of VEGF on spermatogenesis is unclear. Recent findings have revealed several inhibitory effects of VEGF on spermatogenesis (11, 12). We investigated the expression of VEGF in human testes with varicocele. To understand the role of VEGF in human testes with varicocele, we have also compared the expression of VEGF with clinical parameters. Thirty patients with left varicocele (subclinical: n ¼ 3; grade 1: n ¼ 9; grade 2: n ¼ 12; grade 3: n ¼ 6) were enrolled in this study (mean age 34.1 5.7; range 22–50). Twenty-three patients (77%) presented with primary and Received June 11, 2007; revised July 30, 2007; accepted August 10, 2007. The authors have declared that no conflict of interest exists. Reprint requests: Koji Shiraishi, M.D., Ph.D., Department of Urology, Ube Industries Central Hospital, 750 Nishikiwa, Ube, Yamaguchi 755-0151, Japan (FAX: þ1-836-51-9252; E-mail: [email protected]).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.08.030
seven (23%) with secondary infertility. Varicocele was diagnosed after a physical examination, duplex, and color Doppler ultrasonography. Testicular volume was determined using punched-out orchidometer. Semen analyses were performed at least three times before varicocelectomy. Total motile sperm count (TM) was calculated by multiplying sperm concentration (106/mL) and semen volume (mL). Mean TM was calculated for each patient. Microsurgical varicocelectomy was performed through subinguinal or inguinal approach under spinal anesthesia. At the end of operation, biopsy samples were obtained from the left testes via a scrotal incision. Eight testicular biopsy specimens from fertile adults aged 23–40 (mean SE; 36.5 6.2) were included as controls (five patients with scrotal hydrocele, three patients who were performed orchiopexy after contralateral testicular torsion). All the physical examinations, varicocelectomies, and biopsies were performed by one of the investigators (K.S.). After the explanation of the purpose of this study, informed consents from all patients and ethical approval for human study were obtained. Frozen specimens were homogenized in 5 to 10 volumes of sucrose–Tris–EGTA buffer (pH 7.4) with protease inhibitors. Equal amounts of protein (15 mg) were electrophoresed on 15% gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dried milk in Tris-buffered saline for 1 hour, the membranes were incubated with rabbit polyclonal VEGF (A-20, at a dilution of 1:500, Santa Cruz Biotech., Santa Cruz, CA) in 1% bovine serum albumin overnight at room temperature. Then the membranes were reacted with secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 1 hour. The antigens were visualized with an ECL Western blotting detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) and quantified using an image analyzer (Densitograph AE-6900M; Atto Co., Tokyo, Japan). The data were calculated as percentage of the control where control values have been assigned a value of 100%. Data were expressed as mean SE. After fixation in Bouin’s solution for 30 minutes, 4-mm paraffin sections were mounted on silan-coated glass slides (Dako Japan, Kyoto, Japan). After inhibition of endogenous
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
1313
peroxidases in hydrogen peroxide solution, antigen was retrieved by heating in citrate buffer (pH 6) at 98 C for 2 5 minutes. After nonspecific binding was blocked by rabbit serum, overnight incubation was performed using the antibodies for VEGF (at a dilution of 1:200). HRP-conjugated goat antirabbit immunoglobulin was used as a secondary antibody (1:100, Dako) for 1 hour and then visualized by the avidin–biotin complex method. Sections were counterstained with hematoxylin. Omitting the first antibody served as a negative control. The Statview J 4.02 program (Abacus Concepts, Inc., Berkeley, CA) was used for statistical analysis. The VEGF expressions in each group were evaluated by the KruskalWallis test. To examine the relationship between VEGF expressions and clinicopathologic parameters, Spearman rank correlation coefficients were performed. A P value < .05 was considered as statistically significant. Representative immunoblot for VEGF (A) and its quantification (B) are shown in Figure 1. In control testis, VEGF expression showed a single band at 21 kDa, indicating constitutive expression of VEGF in normal testis. In patients with grade 2 or 3 varicocele (n ¼ 18), the expression was significantly higher (2.4-fold) than those of control (n ¼ 8) or
subclinical/grade 1 varicocele (n ¼ 12) (p< .01). In control testis, VEGF was present in the cytosol of Leydig cells (C, 400). There was no immunoreactivity for blood vessels, peritubular cells, and intratubular components. As well as an increased immunoreactivity for Leydig cells, Sertoli cells were also positive for VEGF in testis with varicocele (D, 400). These localizations were evident in testes with grade 2 or 3 varicocele. There was no apparent staining in negative control sections. There were significant inverse correlations between VEGF levels and TM (P<.05, r ¼ 0.335) or testicular volume (P<.05, r ¼ 0.286). No significant correlation was observed about age and serum hormonal parameters (LH, FSH, and testosterone). Our results indicate that the expression of VEGF was increased in testes with varicocele, consistent with the previous finding using rat varicocele model (2). The increased expression was inversely correlated with TM and testicular volume, indicating that VEGF has several harmful effects on spermatogenesis in testes with varicocele. A number of factors stimulating human VEGF gene expression have been identified, including cAMP, steroid hormones, hypoxia, glucose, cobalt, growth factors, and cytokines (13). Given the facts that systemic hypoxia
FIGURE 1 Representative VEGF immunoblots from control and varicocele patients (A). Quantification of VEGF expressed as percent of control (B). Data are expressed as mean SE (*P< .01). Representative VEGF immunohistochemistry in control (C) and varicoele (D) testes (400). Immunoreactivity in Leydig cells was increased in testes with varicocele. Arrows indicate the immunoreactivity in Sertoli cells. Sub ¼ subclinical; G1 ¼ grade 1; G2 ¼ Grade 2; G3 ¼ Grade 3.
Shiraishi. Varicocele and VEGF. Fertil Steril 2008.
1314
Shiraishi and Naito
Correspondence
Vol. 90, No. 4, October 2008
increased the expression of VEGF (up to threefold) in mouse testes (14) and hypoxia is supposed to exist in testes with varicocele (1), increased expression of VEGF may occur in response to hypoxic stimulation. In fact, Kilinc et al. (2) observed the increased expressions of hypoxia-inducible factor-1a and VEGF after experimental varicocele of rats. The internal spermatic vein with varicocele has also shown to be exposed to hypoxia by increased expression of HIF-1a (3). Several possibilities are raised to explain the inverse correlations between VEGF expression and spermatogenesis. First, VEGF targets not only endothelial cells but also nonendothelial cells. An overexpression of VEGF in the testis suppresses the spermatogenesis by inhibiting the spermatogonial proliferation (12) and resulting in aspermatogenesis and infertility (11). Second, VEGF directly acts on the angiogenesis from the preexisting vasculature through complex interactions among endothelial cells, basement membrane, and extracellular matrix, resulting in a relative increase of interstitial tissue over seminiferous tubules (15). In fact, an increased amount of interstitial tissue (e.g., proliferation of Leydig cells, thickening of tubular basement membrane, increased deposition of fibrous tissues) have been observed in testes with varicocele (16). Hypoxia stimulates Leydig cell proliferation via an increase of VEGF production in mouse Leydig cells (17), resulting in a relative increase of interstitial elements. Third, recent studies have demonstrated that VEGF causes endothelial nitric oxide synthase phosphorylation, and result in endothelial nitric oxide (NO) release through the phosphatidylinositol 30 -kinase-AKTdependent pathway in vascular endothelial cells (18). Taken together, the results that endothelial NADPH-diaphorase activity, which is a marker of NO production, was increased in testes with varicocele (19), and was involved in the increasing size of varicocele, this NO-mediated indirect action of VEGF might be involved in the increasing size of varicocele (Fig. 1). Intense expression of VEGF in Leydig cells (Fig. 1) is in agreement with the result reported by Ergun et al. (9) using human testes. Gonadotropin-induced production of VEGF from rat and mouse Leydig cells has been reported in vivo (20) and in vitro (21, 22). Experimental varicocele of rats induced an increase in testicular permeability, which was enhanced by treatment with hCG. (23). Thus, Leydig cells might play a role to regulate the angiogenesis and local vascular permeability. We did not detect strong immunoreactivity for VEGF in Sertoli cells in the control testes (Fig. 1). Testes from different ages of patients might be one reason to explain the difference from the results reported by Ergun et al. (9). A though understanding of VEGF’s role in the testis will shed important insight into pathogenesis of varicocele and the other testicular disorders as well. This result proFertility and Sterility
motes a possible therapeutic strategy aimed at blocking VEGF with or without conventional therapies against varicocele. Koji Shiraishi, M.D., Ph.D. Katsusuke Naito, M.D., Ph.D. Department of Urology, Yamaguchi University School of Medicine, Yamaguchi, Japan REFERENCES 1. Chakraborty J, Hikim AP, Jhunjhunwala JS. Stagnation of blood in the microcirculatory vessels in the testes of men with varicocele. J Androl 1985;6:117–26. 2. Kilinc F, Kayaselcuk F, Aygun C, Guvel S, Egilmez T, Ozkardes H. Experimental varicocele induces hypoxia inducible factor-1alpha, vascular endothelial growth factor expression and angiogenesis in the rat testis. J Urol 2004;172:1188–91. 3. Lee J-D, Jeng S-Y, Lee T-H. Increased expression of hypoxia-inducible factor-1a in the internal spermatic vein of patients with varicocele. J Urol 2006;175:1045–8. 4. Nagler HM, Lizza E, House SD, Tomashesky P, Lopowsky HH. Testicular hemodynamic changes after the surgical creation of a varicocele in the rat intravital microscopic observations. J Androl 1987;8:292–8. 5. Conn G, Soderman DD, Schaeffer MT, Wile M, Hatcher VB, Thomas KA. Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line. Proc Natl Acad Sci USA 1990;87:1323–7. 6. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25. 7. Bott RC, McFee RM, Clopton DT, Toombs C, Cupp AS. Vascular endothelial growth factor and kinase domain region receptor are involved in both seminiferous cord formation and vascular development during testis morphogenesis in the rat. Biol Repord 2006;75:56–67. 8. Brown LF, Yeo KT, Berse B, Morgentaler A, Dvorak HF, Rosen S. Vascular permeability factor (vascular endothelial growth factor) is strongly expressed in the normal male genital tract and is present in substantial quantities in semen. J Urol 1995;154:576–9. 9. Ergun S, Kilic N, Fiedler W, Mukhopadhyay AK. Vascular endothelial growth factor and it receptors in normal human testicular tissue. Mol Cell Endocrinol 1997;131:9–20. 10. Ergun S, Luttmer W, Fiedler W, Holstein AF. Functional expression and localization of vascular endothelial growth factor and its receptors in the human epididymis. Biol Reprod 1998;58:160–8. 11. Korpelainen EI, Karkkainen MJ, Tenhunen A, Lakso M, Rauvala H, Vierula M, et al. Overexpression of VEGF in testis and epididymis causes infertility in transgenic mice: evidence for non-endothelial targets for VEGF. J Cell Biol 1998;143:1705–12. 12. Nalbandian A, Dettin L, Dym M, Ravindranath N. Expression of vascular endothelial growth factor receptors during male germ cell differentiation in the mouse. Biol Reprod 2003;69:985–94. 13. Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989;84:1470–8. 14. Marti HH, Risau W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factors and its receptors. Proc Natl Acad Sci USA 1998;95:15809–14. 15. Korff T, Augustin HG. Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J Cell Sci 1999;112:3249–58. 16. Abdelrahim F, Mostafa A, Hamdy A, Mabrouk M, el-Kholy M, Hassan O. Testicular morphology and function in varicocele patients: pre-operative and post-operative histopathology. Br J Urol 1993;72:643–7. 17. Hwang GS, Wang SW, Tseng WM, Yu CH, Wang PS. Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells. Am J Physiol Endocrinol Metab 2007;292: E1763–9.
1315
18. Michell BJ, Grifiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tigains T, Bozinovski S, et al. The AKT kinase signals directly to endothelial nitric oxide synthase. Curr Biol 1999;9:845–8. 19. Shiraishi K, Naito K. Nitric oxide produced in the testis is involved in dilatation of the internal spermatic vein that compromises spermatogenesis in infertile men with varicocele. BJU Int 2007;99: 1086–90. 20. Rudolfsson SH, Wikstrom P, Jonsson A, Collin O, Bergh A. Hormonal regulation and functional role of vascular endothelial growth factor A in the rat testis. Biol Reprod 2004;70:340–7.
1316
Shiraishi and Naito
Correspondence
21. Anand RJK, Paust H-J, Altenpohl K, Mukhopadhyay AK. Regulation of vascular endothelial growth factor production by Leydig cells in vitro: the role of protein kinase A and mitogen-activated protein kinase cascade. Biol Reprod 2003;68:1663–73. 22. Schwarzenbach H, Chakrabarti G, Joachim Paust H, Mukhopadhyay AK. Gonadotropin-mediated regulation of the murine VEGF expression in MA-10 Leydig cells. J Androl 2004;25:128–39. 23. Salama N, Bergh A, Damber J-E. The changes in testicular vascular permeability during progression of the experimental varicocele. Eur Urol 2003;43:84–91.
Vol. 90, No. 4, October 2008
Several mechanisms have been postulated to explain varicocele-induced deterioration of spermatogenesis. Hypoxia is known to be one of the factors involved in the pathophysiology of varicocele (1–3). Varicocele also alters testicular microcirculation to maintain adequate microenvironments in respond to hypoxia for spermatogenesis and steroidogenesis (4). However, few investigations have been done to examine the molecular events to address this issue. Vascular endothelial growth factor (VEGF) is a 21-kDa glycoprotein with a specific mitogenic effect for vascular endothelial cells (5), and is a major regulator of endothelial cell proliferation, angiogenesis, and vascular permeability (6). Vascular endothelial growth factor has a crucial role during testis morphogenesis through neurovascularization and cord formation (7). In human testis, VEGF has been shown to be expressed in the male genital tract including Leydig and Sertoli cells, certain epithelial and peritubular cells of the epididymis, and the epithelium of the prostate and seminal vesicle (8–10), and it exerts its biologic function by binding to tyrosine kinase receptor 1 (flt-1) and 2 (flk/KDR), which are located on the surface of endothelial cells, Leydig, and Sertoli cells (9). Increased expression of VEGF in testis has been reported in experimental varicocele using rats (2); however, the role of VEGF on spermatogenesis is unclear. Recent findings have revealed several inhibitory effects of VEGF on spermatogenesis (11, 12). We investigated the expression of VEGF in human testes with varicocele. To understand the role of VEGF in human testes with varicocele, we have also compared the expression of VEGF with clinical parameters. Thirty patients with left varicocele (subclinical: n ¼ 3; grade 1: n ¼ 9; grade 2: n ¼ 12; grade 3: n ¼ 6) were enrolled in this study (mean age 34.1 5.7; range 22–50). Twenty-three patients (77%) presented with primary and Received June 11, 2007; revised July 30, 2007; accepted August 10, 2007. The authors have declared that no conflict of interest exists. Reprint requests: Koji Shiraishi, M.D., Ph.D., Department of Urology, Ube Industries Central Hospital, 750 Nishikiwa, Ube, Yamaguchi 755-0151, Japan (FAX: þ1-836-51-9252; E-mail: [email protected]).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.08.030
seven (23%) with secondary infertility. Varicocele was diagnosed after a physical examination, duplex, and color Doppler ultrasonography. Testicular volume was determined using punched-out orchidometer. Semen analyses were performed at least three times before varicocelectomy. Total motile sperm count (TM) was calculated by multiplying sperm concentration (106/mL) and semen volume (mL). Mean TM was calculated for each patient. Microsurgical varicocelectomy was performed through subinguinal or inguinal approach under spinal anesthesia. At the end of operation, biopsy samples were obtained from the left testes via a scrotal incision. Eight testicular biopsy specimens from fertile adults aged 23–40 (mean SE; 36.5 6.2) were included as controls (five patients with scrotal hydrocele, three patients who were performed orchiopexy after contralateral testicular torsion). All the physical examinations, varicocelectomies, and biopsies were performed by one of the investigators (K.S.). After the explanation of the purpose of this study, informed consents from all patients and ethical approval for human study were obtained. Frozen specimens were homogenized in 5 to 10 volumes of sucrose–Tris–EGTA buffer (pH 7.4) with protease inhibitors. Equal amounts of protein (15 mg) were electrophoresed on 15% gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dried milk in Tris-buffered saline for 1 hour, the membranes were incubated with rabbit polyclonal VEGF (A-20, at a dilution of 1:500, Santa Cruz Biotech., Santa Cruz, CA) in 1% bovine serum albumin overnight at room temperature. Then the membranes were reacted with secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 1 hour. The antigens were visualized with an ECL Western blotting detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) and quantified using an image analyzer (Densitograph AE-6900M; Atto Co., Tokyo, Japan). The data were calculated as percentage of the control where control values have been assigned a value of 100%. Data were expressed as mean SE. After fixation in Bouin’s solution for 30 minutes, 4-mm paraffin sections were mounted on silan-coated glass slides (Dako Japan, Kyoto, Japan). After inhibition of endogenous
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
1313
peroxidases in hydrogen peroxide solution, antigen was retrieved by heating in citrate buffer (pH 6) at 98 C for 2 5 minutes. After nonspecific binding was blocked by rabbit serum, overnight incubation was performed using the antibodies for VEGF (at a dilution of 1:200). HRP-conjugated goat antirabbit immunoglobulin was used as a secondary antibody (1:100, Dako) for 1 hour and then visualized by the avidin–biotin complex method. Sections were counterstained with hematoxylin. Omitting the first antibody served as a negative control. The Statview J 4.02 program (Abacus Concepts, Inc., Berkeley, CA) was used for statistical analysis. The VEGF expressions in each group were evaluated by the KruskalWallis test. To examine the relationship between VEGF expressions and clinicopathologic parameters, Spearman rank correlation coefficients were performed. A P value < .05 was considered as statistically significant. Representative immunoblot for VEGF (A) and its quantification (B) are shown in Figure 1. In control testis, VEGF expression showed a single band at 21 kDa, indicating constitutive expression of VEGF in normal testis. In patients with grade 2 or 3 varicocele (n ¼ 18), the expression was significantly higher (2.4-fold) than those of control (n ¼ 8) or
subclinical/grade 1 varicocele (n ¼ 12) (p< .01). In control testis, VEGF was present in the cytosol of Leydig cells (C, 400). There was no immunoreactivity for blood vessels, peritubular cells, and intratubular components. As well as an increased immunoreactivity for Leydig cells, Sertoli cells were also positive for VEGF in testis with varicocele (D, 400). These localizations were evident in testes with grade 2 or 3 varicocele. There was no apparent staining in negative control sections. There were significant inverse correlations between VEGF levels and TM (P<.05, r ¼ 0.335) or testicular volume (P<.05, r ¼ 0.286). No significant correlation was observed about age and serum hormonal parameters (LH, FSH, and testosterone). Our results indicate that the expression of VEGF was increased in testes with varicocele, consistent with the previous finding using rat varicocele model (2). The increased expression was inversely correlated with TM and testicular volume, indicating that VEGF has several harmful effects on spermatogenesis in testes with varicocele. A number of factors stimulating human VEGF gene expression have been identified, including cAMP, steroid hormones, hypoxia, glucose, cobalt, growth factors, and cytokines (13). Given the facts that systemic hypoxia
FIGURE 1 Representative VEGF immunoblots from control and varicocele patients (A). Quantification of VEGF expressed as percent of control (B). Data are expressed as mean SE (*P< .01). Representative VEGF immunohistochemistry in control (C) and varicoele (D) testes (400). Immunoreactivity in Leydig cells was increased in testes with varicocele. Arrows indicate the immunoreactivity in Sertoli cells. Sub ¼ subclinical; G1 ¼ grade 1; G2 ¼ Grade 2; G3 ¼ Grade 3.
Shiraishi. Varicocele and VEGF. Fertil Steril 2008.
1314
Shiraishi and Naito
Correspondence
Vol. 90, No. 4, October 2008
increased the expression of VEGF (up to threefold) in mouse testes (14) and hypoxia is supposed to exist in testes with varicocele (1), increased expression of VEGF may occur in response to hypoxic stimulation. In fact, Kilinc et al. (2) observed the increased expressions of hypoxia-inducible factor-1a and VEGF after experimental varicocele of rats. The internal spermatic vein with varicocele has also shown to be exposed to hypoxia by increased expression of HIF-1a (3). Several possibilities are raised to explain the inverse correlations between VEGF expression and spermatogenesis. First, VEGF targets not only endothelial cells but also nonendothelial cells. An overexpression of VEGF in the testis suppresses the spermatogenesis by inhibiting the spermatogonial proliferation (12) and resulting in aspermatogenesis and infertility (11). Second, VEGF directly acts on the angiogenesis from the preexisting vasculature through complex interactions among endothelial cells, basement membrane, and extracellular matrix, resulting in a relative increase of interstitial tissue over seminiferous tubules (15). In fact, an increased amount of interstitial tissue (e.g., proliferation of Leydig cells, thickening of tubular basement membrane, increased deposition of fibrous tissues) have been observed in testes with varicocele (16). Hypoxia stimulates Leydig cell proliferation via an increase of VEGF production in mouse Leydig cells (17), resulting in a relative increase of interstitial elements. Third, recent studies have demonstrated that VEGF causes endothelial nitric oxide synthase phosphorylation, and result in endothelial nitric oxide (NO) release through the phosphatidylinositol 30 -kinase-AKTdependent pathway in vascular endothelial cells (18). Taken together, the results that endothelial NADPH-diaphorase activity, which is a marker of NO production, was increased in testes with varicocele (19), and was involved in the increasing size of varicocele, this NO-mediated indirect action of VEGF might be involved in the increasing size of varicocele (Fig. 1). Intense expression of VEGF in Leydig cells (Fig. 1) is in agreement with the result reported by Ergun et al. (9) using human testes. Gonadotropin-induced production of VEGF from rat and mouse Leydig cells has been reported in vivo (20) and in vitro (21, 22). Experimental varicocele of rats induced an increase in testicular permeability, which was enhanced by treatment with hCG. (23). Thus, Leydig cells might play a role to regulate the angiogenesis and local vascular permeability. We did not detect strong immunoreactivity for VEGF in Sertoli cells in the control testes (Fig. 1). Testes from different ages of patients might be one reason to explain the difference from the results reported by Ergun et al. (9). A though understanding of VEGF’s role in the testis will shed important insight into pathogenesis of varicocele and the other testicular disorders as well. This result proFertility and Sterility
motes a possible therapeutic strategy aimed at blocking VEGF with or without conventional therapies against varicocele. Koji Shiraishi, M.D., Ph.D. Katsusuke Naito, M.D., Ph.D. Department of Urology, Yamaguchi University School of Medicine, Yamaguchi, Japan REFERENCES 1. Chakraborty J, Hikim AP, Jhunjhunwala JS. Stagnation of blood in the microcirculatory vessels in the testes of men with varicocele. J Androl 1985;6:117–26. 2. Kilinc F, Kayaselcuk F, Aygun C, Guvel S, Egilmez T, Ozkardes H. Experimental varicocele induces hypoxia inducible factor-1alpha, vascular endothelial growth factor expression and angiogenesis in the rat testis. J Urol 2004;172:1188–91. 3. Lee J-D, Jeng S-Y, Lee T-H. Increased expression of hypoxia-inducible factor-1a in the internal spermatic vein of patients with varicocele. J Urol 2006;175:1045–8. 4. Nagler HM, Lizza E, House SD, Tomashesky P, Lopowsky HH. Testicular hemodynamic changes after the surgical creation of a varicocele in the rat intravital microscopic observations. J Androl 1987;8:292–8. 5. Conn G, Soderman DD, Schaeffer MT, Wile M, Hatcher VB, Thomas KA. Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line. Proc Natl Acad Sci USA 1990;87:1323–7. 6. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4–25. 7. Bott RC, McFee RM, Clopton DT, Toombs C, Cupp AS. Vascular endothelial growth factor and kinase domain region receptor are involved in both seminiferous cord formation and vascular development during testis morphogenesis in the rat. Biol Repord 2006;75:56–67. 8. Brown LF, Yeo KT, Berse B, Morgentaler A, Dvorak HF, Rosen S. Vascular permeability factor (vascular endothelial growth factor) is strongly expressed in the normal male genital tract and is present in substantial quantities in semen. J Urol 1995;154:576–9. 9. Ergun S, Kilic N, Fiedler W, Mukhopadhyay AK. Vascular endothelial growth factor and it receptors in normal human testicular tissue. Mol Cell Endocrinol 1997;131:9–20. 10. Ergun S, Luttmer W, Fiedler W, Holstein AF. Functional expression and localization of vascular endothelial growth factor and its receptors in the human epididymis. Biol Reprod 1998;58:160–8. 11. Korpelainen EI, Karkkainen MJ, Tenhunen A, Lakso M, Rauvala H, Vierula M, et al. Overexpression of VEGF in testis and epididymis causes infertility in transgenic mice: evidence for non-endothelial targets for VEGF. J Cell Biol 1998;143:1705–12. 12. Nalbandian A, Dettin L, Dym M, Ravindranath N. Expression of vascular endothelial growth factor receptors during male germ cell differentiation in the mouse. Biol Reprod 2003;69:985–94. 13. Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989;84:1470–8. 14. Marti HH, Risau W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factors and its receptors. Proc Natl Acad Sci USA 1998;95:15809–14. 15. Korff T, Augustin HG. Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J Cell Sci 1999;112:3249–58. 16. Abdelrahim F, Mostafa A, Hamdy A, Mabrouk M, el-Kholy M, Hassan O. Testicular morphology and function in varicocele patients: pre-operative and post-operative histopathology. Br J Urol 1993;72:643–7. 17. Hwang GS, Wang SW, Tseng WM, Yu CH, Wang PS. Effect of hypoxia on the release of vascular endothelial growth factor and testosterone in mouse TM3 Leydig cells. Am J Physiol Endocrinol Metab 2007;292: E1763–9.
1315
18. Michell BJ, Grifiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tigains T, Bozinovski S, et al. The AKT kinase signals directly to endothelial nitric oxide synthase. Curr Biol 1999;9:845–8. 19. Shiraishi K, Naito K. Nitric oxide produced in the testis is involved in dilatation of the internal spermatic vein that compromises spermatogenesis in infertile men with varicocele. BJU Int 2007;99: 1086–90. 20. Rudolfsson SH, Wikstrom P, Jonsson A, Collin O, Bergh A. Hormonal regulation and functional role of vascular endothelial growth factor A in the rat testis. Biol Reprod 2004;70:340–7.
1316
Shiraishi and Naito
Correspondence
21. Anand RJK, Paust H-J, Altenpohl K, Mukhopadhyay AK. Regulation of vascular endothelial growth factor production by Leydig cells in vitro: the role of protein kinase A and mitogen-activated protein kinase cascade. Biol Reprod 2003;68:1663–73. 22. Schwarzenbach H, Chakrabarti G, Joachim Paust H, Mukhopadhyay AK. Gonadotropin-mediated regulation of the murine VEGF expression in MA-10 Leydig cells. J Androl 2004;25:128–39. 23. Salama N, Bergh A, Damber J-E. The changes in testicular vascular permeability during progression of the experimental varicocele. Eur Urol 2003;43:84–91.
Vol. 90, No. 4, October 2008