- Email: [email protected]
Transient receptor potential vanilloid receptor-1 confers heat resistance to male germ cells
Transient receptor potential vanilloid receptor-1 confers heat resistance to male germ cells Testicular hyperthermia in mice lacking transient receptor potential vanilloid receptor-1 results in a much more rapid and massive germ cell depletion from the seminiferous tubules than in wild-type animals, indicating that this receptor protects germ cells against heat stress. (Fertil Steril 2008;90:1290–3. 2008 by American Society for Reproductive Medicine.)
Transient receptor potential vanilloid receptor-1 (TRPV1) is a ligand-gated, nonselective cation channel that initially was found to be expressed in small- to medium-diameter neurons in dorsal root, trigeminal, and nodose ganglia (1). Subsequent studies have demonstrated that TRPV1 is expressed in many other neuronal and non-neuronal cells, including urothelial cells, cardiomyocytes, gastric epithelial cells, mast cells, thymocytes, and keratinocytes (2–8). Transient receptor potential vanilloid receptor-1 can be activated by vanilloid compounds such as capsaicin, as well as by a number of other ligands that include protons, products of the arachidonic metabolism, and monovalent and divalent cations (9–14). In addition, TRPV1 activity can be influenced by ambient temperature; a slight warming above room temperature (e.g., 37 C) potentiates the responsiveness of TRPV1 to its chemical agonists, and at temperatures of >42 C, this receptor is activated in the absence of exogenous chemical ligands (15). Activation of TRPV1 on neurons results in excitation leading to pain perception, generally referred as nociception, followed by desensitization of the neuron (1). In nonneuronal cells, the effect of activation of TRPV1 usually is either cell protection or apoptosis (2–8). The messenger RNA for TRPV1 has been found in human seminiferous tubules and in rat testes (2). Recently, we demonstrated that TRPV1 protein is expressed by male rat germ cells (Mizrak SC, Gadella BM, van Pelt AMM, van Dissel-Emiliani FMF, unpublished observations). Transient receptor potential vanilloid receptor-1 mostly was found in premeiotic germ cells, whereas spermatocytes only weakly expressed this receptor, and no expression was found on postmeiotic germ cells. This observation is especially intriguing because TRPV1 is a heat transducer, and spermatocytes and spermatids are highly sensitive to heat stress, whereas spermatogonia are much less so. Here, we applied a genetic approach to define the role of TRPV1 in spermatogenesis by using mice lackReceived July 26, 2007; revised and accepted October 22, 2007. Reprint requests: Sefika Canan Mizrak, Fertility Laboratory, Center for Reproductive Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (FAX: 31-20-6977963; E-mail: [email protected]).
1290
ing TRPV1 and analyzed the effects of heat stress on their germ cells. Male mice (4 knockouts, C57BL/6-B6.129S4-Vr1tm1Jul, TRPV1–/– and 4 matching controls, C57BL/6, WT) were obtained at 8 weeks of age from the Jackson Laboratory (Bar Harbor, ME). They were maintained in a temperatureand humidity-controlled room on a 12:12-hour light–dark cycle. At 10 to 11 weeks of age, mice were weighed and exposed to a single heat shock, as described elsewhere (16–18). The experiments were approved by the ethical and animal care board of the University of Utrecht. Briefly, mice were anesthetized, and their scrota were immersed in a thermostatically controlled water bath of 33 C or 43 C for a period of 15 minutes. Seven days after heat exposure, the animals were weighted again and then killed by CO2 inhalation. The testes were then dissected and the epididymes removed. After the testes were weighed, they were immersion fixed in Bouin’s solution, paraffin embedded, sectioned, and either stained with periodic acid-Schiff–hematoxylin or processed for terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate-biotin nick end labeling (TUNEL) to specifically monitor apoptosis of the germ cells, as described elsewhere (19). For the last, testis sections were boiled for 5 minutes in 10 mM citric buffer (pH 6.0) at 98 C and slowly were cooled to room temperature. Endogenous peroxidase was blocked with 3% H2O2 in Milli-Q (Millipore Corporation, Billerica, MA) water for 5 minutes. Sections were washed three times with phosphate-buffered saline before 60-minute incubation in TUNEL mix at 37 C. The TUNEL mix consisted of 0.3 U/mL calf thymus terminal deoxynucleotidyl transferase (Amersham Biosciences, Freiburg, Germany) and 6.66 mM/mL biotin-16,20 -deoxy-uridine50 -triphosphate (Roche, Basel, Switzerland) in terminal transferase buffer (Amersham Biosciences). The TUNEL reaction was stopped by incubation in 300 mM NaCl and 30 mM sodium citrate in Milli Q water for 15 minutes at room temperature. After washing in phosphate-buffered saline, sections were blocked with 2% BSA in phosphate-buffered saline at room temperature for 10 minutes. Sections were treated for 30 minutes at 37 C in a moist chamber with a 1:20 dilution of ExtrAvidin peroxidase-labeled antibody
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.10.081
FIGURE 1 Photomicrographs of sections through mice testes (A, B, C57BL/6, WT; C, D, C57BL/6-B6.129S4-Vr1tm1Jul, TRPV1–/–) heated during 15 minutes at 43 C and processed a week later. (A, C) Periodic acid-Schiff–hematoxylin staining. (B, D) TUNEL labeling. Brown cells, TUNEL (þ) germ cells. Note the number of apoptotic germ cells in the WT testis (diaminobenzidine precipitate) and the lack of these cells in the TRPV1–/– testis. (E) C57BL/6,WT and (F) C57BL/6-B6.129S4-Vr1tm1Jul, TRPV1–/– mice testes heated during 15 minutes at 33 C and processed a week later; both E and F show PAS-hematoxylin staining. Bars, 100 mm.
Mizrak. Function of TRPV1 in spermatogenesis. Fertil Steril 2008.
(E2886, Sigma, St. Louis, MO). After three washes in PBS, detection was performed with diaminobenzidine. Sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted with Pertex (CellPath plc, Hemel Hempstead, UK). The findings in the WT mice were similar to those described elsewhere (20). Testicular weight loss and degeneration of germ cells was observed 1 week after treatment (mean SD, 96 9 mg and 48 9 mg before and 7 days Fertility and Sterility
after hyperthermia, respectively; Fig. 1A). We selected a period of 7 days after hyperthermia exposure because apoptosis was expected to be clearly visible at that time point, and testicular regression should still not have been completed (16–18). Many apoptotic germ cells indeed were observed after heat exposure, as determined by TUNEL labeling of the testis sections (Fig. 1B). Body weights remained unchanged during treatment (29.3 1.4 g and 27.9 1.3 g, before and 7 days after hyperthermia, respectively).
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Local hyperthermia caused a decrease in testicular weight in the TRPV1–/– mice that was similar to that observed in the WT mice (TRPV1–/– mice: 102 4 mg and 45 6 mg, before and 7 days after hyperthermia, respectively, vs. WT mice: 96 9 mg and 48 9 mg, before and 7 days after hyperthermia, respectively), whereas body weight remained unchanged (32.1 0.9 g and 31.3 0.8 g, before and 7 days after hyperthermia, respectively). At the histological level, however, the effect of hyperthermia on the seminiferous epithelium was much more severe in the TRPV1–/– mice as compared with in the WT mice. The seminiferous tubules showed a vacuolated appearance with an almost total absence of germ cells, including spermatogonia, a morphology resembling Sertoli cell–only syndrome (Fig. 1B). In addition and in agreement with the histological observation, almost no TUNEL-labeled cells were found in these mice. It appears that all germ cells had become much more heat sensitive in the absence of TRPV1 and that apoptosis was taking place more rapidly than in WT mice. Treatment at 33 C did not affect spermatogenesis of either the WT or TRPV1–/– mice (Fig. 1E and F). It is known that elevated testicular temperature caused by cryptorchidism or experimental hyperthermia results in testicular regression, as a result of germ cell apoptosis, and in a temporary period of partial or complete infertility (21). Spermatocytes and spermatids are the first cell types to undergo apoptosis after heat shock. Spermatogonia are known to be more heat resistant. In fact, hyperthermia, as performed in this study, has been used to successfully isolate spermatogonial stem cells from mice (18). In view of the present results, it may be concluded that TRPV1 is crucial in preventing spermatogonia from undergoing massive cell death under heat stress. In addition, TRPV1 appears to be important for the kinetics of postmeiotic germ cell depletion after hyperthermia. Activation of TRPV1 thus may be considered a protective mechanism for germ cells when they are exposed to noxious heat. It is interesting to note that spermatogonia express TRPV1, at least in the rat. The lack of this receptor in the TRPV1–/– mice thus may directly affect survival of these germ cells under stress conditions. Because spermatocytes and spermatids do not express TRPV1 in that species, their accelerated disappearance from the heated seminiferous tubules of the TRPV1/ mice may be an indirect effect caused by alterations of other cell populations or factors within the testis. However, TRPV1 expression studies in the mouse testis still need to be performed. The TRPV1–/– mice are known to be developmentally normal and fertile (22). Furthermore, TRPV1–/– mice do not differ from WT mice in their thermoregulatory capacity (23, 24). It therefore is unlikely that the observed effect is due to a more generic response of these mice to heat stress. It is possible, though, that other members of the TRP channels family (25–27) are expressed by the testicular cells and that a compensatory up-regulation of a member or members
1292
Mizrak and van Dissel-Emiliani
Correspondence
of this family has occurred in the TRPV1/ mice. This would perhaps confer greater heat sensitivity to the germ cells. However, this requires further investigation. In conclusion, in this study, we demonstrated that TRPV1 plays a crucial role in the defense of testis against heat stress. Thermosensory channels and their functions have not been studied so far within the context of germ cell physiology. Our observations make it clear that this field of research certainly necessitates further consideration. Sefika Canan Mizraka,b Federica M. F. van Dissel-Emilianib a Fertility Laboratory, Center for Reproductive Medicine, Academic Medical Center, Amsterdam; and b Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands REFERENCES 1. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816–24. 2. Birder LA, Kanai AJ, de Groat WC, et al. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci USA 2001;98:13396–401. 3. Dvorakova M, Kummer W. Transient expression of vanilloid receptor subtype 1 in rat cardiomyocytes during development. Histochem Cell Biol 2001;116:223–5. 4. Kato S, Aihara E, Nakamura A, Xin H, Matsui H. Kohama K, vanilloid receptors in rat gastric epithelial cells: role in cellular protection. Biochem Pharmacol 2003;66:1115–21. 5. Biro T, Maurer M, Modarres S, Lewin NE, Brodie C, Acs G, et al. Characterization of functional vanilloid receptors expressed by mast cells. Blood 1998;91:1332–40. 6. Amantini C, Mosca M, Lucciarini R, Perfumi M, Morrone S, Piccoli M, et al. Distinct thymocyte subsets express the vanilloid receptor VR1 that mediates capsaicin-induced apoptotic cell death. Cell Death Differ 2004;11:1342–56. 7. Denda M, Fuziwara S, Inoue K, Denda S, Akamatsu H, Tomitaka A, et al. Immunoreactivity of VR1 on epidermal keratinocyte of human skin. Biochem Biophys Res Commun 2001;285:1250–2. 8. Kido MA, Muroya H, Yamaza T, Terada Y, Tanaka T. Vanilloid receptor expression in the rat tongue and palate. J Dent Res 2003;82: 393–7. 9. Ahern GP, Brooks IM, Miyares RL, Wang XB. Extracellular cations sensitize and gate capsaicin receptor TRPV1 modulating pain signaling. J Neurosci 2005;25:5109–16. 10. Ferrer-Montiel A, Garcia-Martinez C, Morenilla-Palao C, GarciaSanz N, Fernandez-Carvajal A, Fernandez-Ballester G, et al. Molecular architecture of the vanilloid receptor. Insights for drug design. Eur J Biochem 2004;271:1820–6. 11. Cortright DN, Szallasi A. Biochemical pharmacology of the vanilloid receptor TRPV1. An update. Eur J Biochem 2004;271:1814–9. 12. Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 2001;24:487–517. 13. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, et al. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21:531–43. 14. Neubert JK, Karai L, Jun JH, Kim HS, Olah Z, Iadarola MJ. Peripherally induced resiniferatoxin analgesia. Pain 2003;104: 219–28. 15. Dhaka A, Viswanath V, Patapoutian A. Trp ion channels and temperature sensation. Annu Rev Neurosci 2006;29:135–61.
Vol. 90, No. 4, October 2008
16. Vera Y, Diaz-Romero M, Rodriguez S, Lue Y, Wang C, Swerdloff RS, et al. Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice. Biol Reprod 2004;70:1534–40. 17. Yamamoto CM, Sinha Hikim AP, Huynh PN, Shapiro B, Lue Y, Salameh WA, et al. Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats, triggered by mild testicular hyperthermia. Biol Reprod 2000;63:1683–90. 18. McLean DJ, Russell LD, Griswold MD. Biological activity and enrichment of spermatogonial stem cells in vitamin A-deficient and hyperthermia-exposed testes from mice based on colonization following germ cell transplantation. Biol Reprod 2002;66:1374–9. 19. Mizrak SC, Renault-Mihara F, Parraga M, Bogerd J, van de Kant HJ, Lopez-Casas PP, et al. Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis. Reproduction 2007;133:743–51. 20. Rockett JC, Mapp FL, Garges JB, Luft JC, Mori C, Dix DJ. Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice. Biol Reprod 2001;65:229–39.
Fertility and Sterility
21. Setchell BP. The Parkes Lecture. Heat and the testis. J Reprod Fertil 1998;114:179–94. 22. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, PetersenZeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. 23. Szelenyi Z, Hummel Z, Szolcsanyi J, Davis JB. Daily body temperature rhythm and heat tolerance in TRPV1 knockout and capsaicin pretreated mice. Eur J Neurosci 2004;19:1421–4. 24. Iida T, Shimizu I, Nealen ML, Campbell A, Caterina M. Attenuated fever response in mice lacking TRPV1. Neurosci Lett 2005;378:28–33. 25. Montell C, Birnbaumer L, Flockerzi V. The TRP channels, a remarkably functional family. Cell 2002;108:595–8. 26. Gunthorpe MJ, Benham CD, Randall A, Davis JB. The diversity in the vanilloid (TRPV) receptor family of ion channels. Trends Pharmacol Sci 2002;23:183–91. 27. Voets T, Droogmans G, Wissenbach U, Janssens A, Flockerzi V, Nilius B. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 2004;430:748–54.
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Transient receptor potential vanilloid receptor-1 (TRPV1) is a ligand-gated, nonselective cation channel that initially was found to be expressed in small- to medium-diameter neurons in dorsal root, trigeminal, and nodose ganglia (1). Subsequent studies have demonstrated that TRPV1 is expressed in many other neuronal and non-neuronal cells, including urothelial cells, cardiomyocytes, gastric epithelial cells, mast cells, thymocytes, and keratinocytes (2–8). Transient receptor potential vanilloid receptor-1 can be activated by vanilloid compounds such as capsaicin, as well as by a number of other ligands that include protons, products of the arachidonic metabolism, and monovalent and divalent cations (9–14). In addition, TRPV1 activity can be influenced by ambient temperature; a slight warming above room temperature (e.g., 37 C) potentiates the responsiveness of TRPV1 to its chemical agonists, and at temperatures of >42 C, this receptor is activated in the absence of exogenous chemical ligands (15). Activation of TRPV1 on neurons results in excitation leading to pain perception, generally referred as nociception, followed by desensitization of the neuron (1). In nonneuronal cells, the effect of activation of TRPV1 usually is either cell protection or apoptosis (2–8). The messenger RNA for TRPV1 has been found in human seminiferous tubules and in rat testes (2). Recently, we demonstrated that TRPV1 protein is expressed by male rat germ cells (Mizrak SC, Gadella BM, van Pelt AMM, van Dissel-Emiliani FMF, unpublished observations). Transient receptor potential vanilloid receptor-1 mostly was found in premeiotic germ cells, whereas spermatocytes only weakly expressed this receptor, and no expression was found on postmeiotic germ cells. This observation is especially intriguing because TRPV1 is a heat transducer, and spermatocytes and spermatids are highly sensitive to heat stress, whereas spermatogonia are much less so. Here, we applied a genetic approach to define the role of TRPV1 in spermatogenesis by using mice lackReceived July 26, 2007; revised and accepted October 22, 2007. Reprint requests: Sefika Canan Mizrak, Fertility Laboratory, Center for Reproductive Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (FAX: 31-20-6977963; E-mail: [email protected]).
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ing TRPV1 and analyzed the effects of heat stress on their germ cells. Male mice (4 knockouts, C57BL/6-B6.129S4-Vr1tm1Jul, TRPV1–/– and 4 matching controls, C57BL/6, WT) were obtained at 8 weeks of age from the Jackson Laboratory (Bar Harbor, ME). They were maintained in a temperatureand humidity-controlled room on a 12:12-hour light–dark cycle. At 10 to 11 weeks of age, mice were weighed and exposed to a single heat shock, as described elsewhere (16–18). The experiments were approved by the ethical and animal care board of the University of Utrecht. Briefly, mice were anesthetized, and their scrota were immersed in a thermostatically controlled water bath of 33 C or 43 C for a period of 15 minutes. Seven days after heat exposure, the animals were weighted again and then killed by CO2 inhalation. The testes were then dissected and the epididymes removed. After the testes were weighed, they were immersion fixed in Bouin’s solution, paraffin embedded, sectioned, and either stained with periodic acid-Schiff–hematoxylin or processed for terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate-biotin nick end labeling (TUNEL) to specifically monitor apoptosis of the germ cells, as described elsewhere (19). For the last, testis sections were boiled for 5 minutes in 10 mM citric buffer (pH 6.0) at 98 C and slowly were cooled to room temperature. Endogenous peroxidase was blocked with 3% H2O2 in Milli-Q (Millipore Corporation, Billerica, MA) water for 5 minutes. Sections were washed three times with phosphate-buffered saline before 60-minute incubation in TUNEL mix at 37 C. The TUNEL mix consisted of 0.3 U/mL calf thymus terminal deoxynucleotidyl transferase (Amersham Biosciences, Freiburg, Germany) and 6.66 mM/mL biotin-16,20 -deoxy-uridine50 -triphosphate (Roche, Basel, Switzerland) in terminal transferase buffer (Amersham Biosciences). The TUNEL reaction was stopped by incubation in 300 mM NaCl and 30 mM sodium citrate in Milli Q water for 15 minutes at room temperature. After washing in phosphate-buffered saline, sections were blocked with 2% BSA in phosphate-buffered saline at room temperature for 10 minutes. Sections were treated for 30 minutes at 37 C in a moist chamber with a 1:20 dilution of ExtrAvidin peroxidase-labeled antibody
Fertility and Sterility Vol. 90, No. 4, October 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.10.081
FIGURE 1 Photomicrographs of sections through mice testes (A, B, C57BL/6, WT; C, D, C57BL/6-B6.129S4-Vr1tm1Jul, TRPV1–/–) heated during 15 minutes at 43 C and processed a week later. (A, C) Periodic acid-Schiff–hematoxylin staining. (B, D) TUNEL labeling. Brown cells, TUNEL (þ) germ cells. Note the number of apoptotic germ cells in the WT testis (diaminobenzidine precipitate) and the lack of these cells in the TRPV1–/– testis. (E) C57BL/6,WT and (F) C57BL/6-B6.129S4-Vr1tm1Jul, TRPV1–/– mice testes heated during 15 minutes at 33 C and processed a week later; both E and F show PAS-hematoxylin staining. Bars, 100 mm.
Mizrak. Function of TRPV1 in spermatogenesis. Fertil Steril 2008.
(E2886, Sigma, St. Louis, MO). After three washes in PBS, detection was performed with diaminobenzidine. Sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted with Pertex (CellPath plc, Hemel Hempstead, UK). The findings in the WT mice were similar to those described elsewhere (20). Testicular weight loss and degeneration of germ cells was observed 1 week after treatment (mean SD, 96 9 mg and 48 9 mg before and 7 days Fertility and Sterility
after hyperthermia, respectively; Fig. 1A). We selected a period of 7 days after hyperthermia exposure because apoptosis was expected to be clearly visible at that time point, and testicular regression should still not have been completed (16–18). Many apoptotic germ cells indeed were observed after heat exposure, as determined by TUNEL labeling of the testis sections (Fig. 1B). Body weights remained unchanged during treatment (29.3 1.4 g and 27.9 1.3 g, before and 7 days after hyperthermia, respectively).
1291
Local hyperthermia caused a decrease in testicular weight in the TRPV1–/– mice that was similar to that observed in the WT mice (TRPV1–/– mice: 102 4 mg and 45 6 mg, before and 7 days after hyperthermia, respectively, vs. WT mice: 96 9 mg and 48 9 mg, before and 7 days after hyperthermia, respectively), whereas body weight remained unchanged (32.1 0.9 g and 31.3 0.8 g, before and 7 days after hyperthermia, respectively). At the histological level, however, the effect of hyperthermia on the seminiferous epithelium was much more severe in the TRPV1–/– mice as compared with in the WT mice. The seminiferous tubules showed a vacuolated appearance with an almost total absence of germ cells, including spermatogonia, a morphology resembling Sertoli cell–only syndrome (Fig. 1B). In addition and in agreement with the histological observation, almost no TUNEL-labeled cells were found in these mice. It appears that all germ cells had become much more heat sensitive in the absence of TRPV1 and that apoptosis was taking place more rapidly than in WT mice. Treatment at 33 C did not affect spermatogenesis of either the WT or TRPV1–/– mice (Fig. 1E and F). It is known that elevated testicular temperature caused by cryptorchidism or experimental hyperthermia results in testicular regression, as a result of germ cell apoptosis, and in a temporary period of partial or complete infertility (21). Spermatocytes and spermatids are the first cell types to undergo apoptosis after heat shock. Spermatogonia are known to be more heat resistant. In fact, hyperthermia, as performed in this study, has been used to successfully isolate spermatogonial stem cells from mice (18). In view of the present results, it may be concluded that TRPV1 is crucial in preventing spermatogonia from undergoing massive cell death under heat stress. In addition, TRPV1 appears to be important for the kinetics of postmeiotic germ cell depletion after hyperthermia. Activation of TRPV1 thus may be considered a protective mechanism for germ cells when they are exposed to noxious heat. It is interesting to note that spermatogonia express TRPV1, at least in the rat. The lack of this receptor in the TRPV1–/– mice thus may directly affect survival of these germ cells under stress conditions. Because spermatocytes and spermatids do not express TRPV1 in that species, their accelerated disappearance from the heated seminiferous tubules of the TRPV1/ mice may be an indirect effect caused by alterations of other cell populations or factors within the testis. However, TRPV1 expression studies in the mouse testis still need to be performed. The TRPV1–/– mice are known to be developmentally normal and fertile (22). Furthermore, TRPV1–/– mice do not differ from WT mice in their thermoregulatory capacity (23, 24). It therefore is unlikely that the observed effect is due to a more generic response of these mice to heat stress. It is possible, though, that other members of the TRP channels family (25–27) are expressed by the testicular cells and that a compensatory up-regulation of a member or members
1292
Mizrak and van Dissel-Emiliani
Correspondence
of this family has occurred in the TRPV1/ mice. This would perhaps confer greater heat sensitivity to the germ cells. However, this requires further investigation. In conclusion, in this study, we demonstrated that TRPV1 plays a crucial role in the defense of testis against heat stress. Thermosensory channels and their functions have not been studied so far within the context of germ cell physiology. Our observations make it clear that this field of research certainly necessitates further consideration. Sefika Canan Mizraka,b Federica M. F. van Dissel-Emilianib a Fertility Laboratory, Center for Reproductive Medicine, Academic Medical Center, Amsterdam; and b Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands REFERENCES 1. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816–24. 2. Birder LA, Kanai AJ, de Groat WC, et al. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci USA 2001;98:13396–401. 3. Dvorakova M, Kummer W. Transient expression of vanilloid receptor subtype 1 in rat cardiomyocytes during development. Histochem Cell Biol 2001;116:223–5. 4. Kato S, Aihara E, Nakamura A, Xin H, Matsui H. Kohama K, vanilloid receptors in rat gastric epithelial cells: role in cellular protection. Biochem Pharmacol 2003;66:1115–21. 5. Biro T, Maurer M, Modarres S, Lewin NE, Brodie C, Acs G, et al. Characterization of functional vanilloid receptors expressed by mast cells. Blood 1998;91:1332–40. 6. Amantini C, Mosca M, Lucciarini R, Perfumi M, Morrone S, Piccoli M, et al. Distinct thymocyte subsets express the vanilloid receptor VR1 that mediates capsaicin-induced apoptotic cell death. Cell Death Differ 2004;11:1342–56. 7. Denda M, Fuziwara S, Inoue K, Denda S, Akamatsu H, Tomitaka A, et al. Immunoreactivity of VR1 on epidermal keratinocyte of human skin. Biochem Biophys Res Commun 2001;285:1250–2. 8. Kido MA, Muroya H, Yamaza T, Terada Y, Tanaka T. Vanilloid receptor expression in the rat tongue and palate. J Dent Res 2003;82: 393–7. 9. Ahern GP, Brooks IM, Miyares RL, Wang XB. Extracellular cations sensitize and gate capsaicin receptor TRPV1 modulating pain signaling. J Neurosci 2005;25:5109–16. 10. Ferrer-Montiel A, Garcia-Martinez C, Morenilla-Palao C, GarciaSanz N, Fernandez-Carvajal A, Fernandez-Ballester G, et al. Molecular architecture of the vanilloid receptor. Insights for drug design. Eur J Biochem 2004;271:1820–6. 11. Cortright DN, Szallasi A. Biochemical pharmacology of the vanilloid receptor TRPV1. An update. Eur J Biochem 2004;271:1814–9. 12. Caterina MJ, Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci 2001;24:487–517. 13. Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, et al. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21:531–43. 14. Neubert JK, Karai L, Jun JH, Kim HS, Olah Z, Iadarola MJ. Peripherally induced resiniferatoxin analgesia. Pain 2003;104: 219–28. 15. Dhaka A, Viswanath V, Patapoutian A. Trp ion channels and temperature sensation. Annu Rev Neurosci 2006;29:135–61.
Vol. 90, No. 4, October 2008
16. Vera Y, Diaz-Romero M, Rodriguez S, Lue Y, Wang C, Swerdloff RS, et al. Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice. Biol Reprod 2004;70:1534–40. 17. Yamamoto CM, Sinha Hikim AP, Huynh PN, Shapiro B, Lue Y, Salameh WA, et al. Redistribution of Bax is an early step in an apoptotic pathway leading to germ cell death in rats, triggered by mild testicular hyperthermia. Biol Reprod 2000;63:1683–90. 18. McLean DJ, Russell LD, Griswold MD. Biological activity and enrichment of spermatogonial stem cells in vitamin A-deficient and hyperthermia-exposed testes from mice based on colonization following germ cell transplantation. Biol Reprod 2002;66:1374–9. 19. Mizrak SC, Renault-Mihara F, Parraga M, Bogerd J, van de Kant HJ, Lopez-Casas PP, et al. Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis. Reproduction 2007;133:743–51. 20. Rockett JC, Mapp FL, Garges JB, Luft JC, Mori C, Dix DJ. Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice. Biol Reprod 2001;65:229–39.
Fertility and Sterility
21. Setchell BP. The Parkes Lecture. Heat and the testis. J Reprod Fertil 1998;114:179–94. 22. Caterina MJ, Leffler A, Malmberg AB, Martin WJ, Trafton J, PetersenZeitz KR, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. 23. Szelenyi Z, Hummel Z, Szolcsanyi J, Davis JB. Daily body temperature rhythm and heat tolerance in TRPV1 knockout and capsaicin pretreated mice. Eur J Neurosci 2004;19:1421–4. 24. Iida T, Shimizu I, Nealen ML, Campbell A, Caterina M. Attenuated fever response in mice lacking TRPV1. Neurosci Lett 2005;378:28–33. 25. Montell C, Birnbaumer L, Flockerzi V. The TRP channels, a remarkably functional family. Cell 2002;108:595–8. 26. Gunthorpe MJ, Benham CD, Randall A, Davis JB. The diversity in the vanilloid (TRPV) receptor family of ion channels. Trends Pharmacol Sci 2002;23:183–91. 27. Voets T, Droogmans G, Wissenbach U, Janssens A, Flockerzi V, Nilius B. The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels. Nature 2004;430:748–54.
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