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Can Vasoactive Intestinal Peptide (VIP) protect ischemiareperfusion injury of testes in prepubertal rats?
722
721 CAN VASOACTIVE INTESTINAL ISCHEMIAREPERFUSION INJURY RATS? Can’,
Tiire
F.‘,
Tunpel
N.*,
PEPTIDE OF TESTES
(VIP) PROTECT IN PREPUBERTAL
Uysal O.j, Gtirer F.‘, Ak D.?, Tunnel M.1
‘Department of Urology, Osmangazi University, Eskisehir, Turkey, ‘Department of Physiology, Osmangazi University. Eskisehir, Turkey, ‘Department of Histology-Embryology, Oamangazi University, Eskisehir, Turkey, Department of Analytical Chemistry, Anadolu University, Turkey INTRODUCTION & OBJECTIVES: Vasoactive intestinal peptide (VIP) is a member of a family of neuropeptides related to the hormone secretin. It is a bioactive peptide with broad biological effects on a widespread distribution in the body including testes. It has been reported that VIP protects many organs and the tissues against injury caused by oxidants and other toxins. In this study we investigated the protective effect of VIP on ischemia-reperfusion injury of the testes in prepubertal rata. MATERIALS & METHODS: Prepubertal (37-45 days old) male SpragueDawley rats were divided into 7 groups. Group I was the sham operated (n: 8). group II (n: 10) underwent testicular torsion for 2 h, groups III (n: 8) and V (n: IO) received saline vehicle and VIP respectively and underwent detortion for I h following 2 h of tortion. group IV (n: IO) and VI (n: 9) received saline vehicle and VIP respectively and underwent detortion for 4 h following 2 h of torsion. Group VII (n: 9) served to determine the basal values. Tortions were created by rotating the right testis 720” in a clockwise direction. Levels of auperoxide dismutase (SOD). catalase and malondialdehyde (MDA) were determined in testicular tissue. Histological evaluation was done in all groups. Difference was assessed using t-test and XL-test for parametric and nonparametric data respectively. RESULTS: In group II, the level of MDA was significantly increased when compared to the control groups (I and VII). The significant histologic tissue injury and mast cell degranulation were also observed between group II and control groups. In groups V and VI (VIP-treated), the levels of SOD and catalase were significantly decreased when compared to untreated groups (III. IV). Specimens from group III had a significantly greater histologic injury than group V. In groups III and IV, degranulation and exocytosis were observed in mast cells while most of the mast cells were granulated in groupe V and VI. The histochemical staining characteristics of mast cells were also different between VIP-treated and untreated groups. CONCLUSION:
VIP can protect testicular tissue from reperfusion
ATP: MORE THAN JUST ROCKET FUEL FOR SPERM? Thompson Cecil’, Mikhaihdis Dimitri’, Morgan Banks Frederick’, Calvert Robert’, Robert’, Burnstock Geoffrey’ ‘Urology, Royal Free Hospital. London, United Kingdom. ‘Chemical Pathology, Royal Free ZAutonomtc Neurowence Institute. Royal Free Hospital. London. United Kingdom, Hospital, London. United Kingdom
INTRODUCTION & OBJECTIVES:
Adenowte 5’.triphosphate (ATP) is known to be an tmportant energy source in sperm providmg fuel for motihty. In additton to its role in intrxellular energy metabolism, ATP i\ known to act as an extracellular rignalling molecule, affectmg the function and dtfferentiatton of various cell types via P2 purinoceptors (I). Testicular sperm are immotile. however motility is gained as they pass front the epididymal head to the tail. The mechanisms underlying this differentiation are unclear. ExpresGon of P2X purtnoceptor~ ha? been reported during various stages of rat \permatogenests. mdicative of a role during dtfferenttation (2). In additton, incubation of sperm with ATP has been found to stgnificantly improve in vitro fertilisation rates (3). The purpose of this study was to examine purinoceptor expression in epidtdymal sperm. and to examine any alteration during the maturation process leading to motility.
MATERIALS & METHODS: Testes were obtamed from euthanised mice, rats and hamsters Human tissue was obtained from appropriately consented patients undergoing orchidectomy for prostate or testicular cancer. Section\ were taken from both the head and tdil of the epididymi\, and placed adjacently on the same slide. lmmunohistochemistry was performed on the fixed sections using primary antibodies to each of the seven P2X receptor \ubtype\. RESULTS: P2XJ and P2X2 receptors were present on sperm contained wtthin the head of the epididymi\ from all species examined. This expression was undetectable on sperm in the epidtdymal tall m all species except for humans tn which xmte expression persisted. P2X3 exprewon was observed on epididymal head, but not tail sperm, from the mouse and the hamster only. P2X4 expression was observed in the mouse and human sperm from both the head and tail of the epidtdymir. P?XS expression was observed to a similar degree in both the head and the tad eptdtdymal sperm from the tnouse. P2X6 and P2X7 staining was not observed in sperm from etther the head or tail of the epidtdymis from any species. In control experiment\, no immunoreactivity was observed when the primary antibody was omttted and was pre-abhorbed. CONCLUSION: Thih study uniquely demonstrates that beveral P2X purmoceptors subtype\ are expressed on epididymal sperm, although there are some species differences. There is strong expression on immature sperm contained within the head of the epididymis, and a wbxquent. but variable loss of expression on more mature sperm contained within the caudal eptdidymis. Sperm undergo significant maturation in the epididymis. Altered sperm expression of purinergic receptorc may be involved in this process. ATP has already been shown to nnprove fertility in vttro (3). Specific targeting of these receptors may lead to therapeutic agents. vvhich might be effective in v’ivo. Reference?, al Cell Tis\
injury.
I. Abbracchio M.P., Burnstock G. Jpn J Pharmacol 358, 139.145 2. Glass R. et Org 2001: 16Y: 377-3X7 3. Ro\sato M. et al Hum Repr lY99:14 (3) 694.hY7.
723 ULTRASTRUCTURE AND PHARMACOLOGY TESTICULAR CAPSULE CONTRACTION Banks Frederick’, Burnstock Geoffrey’ ‘Urology. Ho\pttal, Hospital,
Calvert
Robert’.
Turmaine
Mark’.
Knight
OF THE Gill’.
Morgan
HUMAN Robert’.
Royal Free Ho\pttal, London, United Kingdom, ‘Anatomy. UmverGty College ‘Autonomic Neumxiencc In\tttute. Royal Free London, United Kingdom, London. United Kingdom
INTRODUCTION & OBJECTIVES: Sperm contained
within the tests\ are immotile. The mechanism by which aperm move front the testis to the epidtdymt? i\ poorly understood. In thi\ study we demonstrate smooth muscle wtthin the human testicular capsule (tunica albuginea). and charactertse tts contractile response. Contraction of the testicular capwlc may ha\e a de in expelling sperm out of the testis into the epidtdymw
MATERIALS & METHODS: Following prior ethical approval and Informed congent. testicular capsular tissue wa\ taken from patients undergoing orchidectomy for either pro\tattc or testicular cancer, or for gender reassignment (n=S). Tiswe for pharmacological evaluation was immediately w\pended m organ baths containing oxygenated Kreb’s solution at 3&“C. Tt~we was pretensioned to Ig and subJected to electrical field \ttmulation (EFS) of autonomic newes at I00 v. 0.3 m\, 30 s ev’ery 5 min. in the frequency range 0.5-32 HI. Following this. exogenom noradrenalme (NA). (single dohe\ IO nmol-0 3 mmol) acctylcholine (ACh) (cumulative doses IO nmol-0.3 mmol) and adeno\ine 5‘ triphosphate (ATP) (single do\e\ nm-0.3 mmol) were added to e\tabh\h conccntrationrrpon\e curve\. Tiwte wxtion\ were examined ustng transmtssion clcctrott microscopy. Further sectton\ underwent immunohi\tological evaluation using c\tabli\hed anti smooth muscle myoatn anttbodiej
IO0
RESULTS: Concentratton dependent contraction was observed to NA. giving an EC50 equivalent to I .hS b,mol. Rcsponaea to nerve ~timulatmn were small and inconsistent and thaw to cxogenoualy applied ACh and ATP were meffective. Immunohi\tological studieh demonstrated small clusters of smooth tmuscle cells throughout the capsule. No obvious pattern or layer formation of smooth muscle was obwwd itt the capsule. Electron micrwcopy demonstrated smooth muxle cell\. apparently not in any eabdy recogniwble at-rangement. although they vvere for the mo\t pnrt arranged longitudmally. Interconnecting proce\\es and gap ,junctions between muscle cells vvere xxm. The neural component wa\ sparse, although. characteristic neuronal vanco\ttte\ containing synaptic v’esicleh vvere observed in tmmedtate proxtmity to smooth muwle cell\. CONCLUSION: Smooth muxlc cell\ are present wtthtn the human testicular capsule. The\e cell\ are re\pon\ivc to NA in a concentration~dependent manner. This suggests that te\ttctdx capsular contractton may be under lympathetic nerv’ou\ system control. Testicular contraction may have a physiological role tn expelling sperm out 01 the testis into the cpididymi\. Stimulation of such contractiott may be relevant in the treatment of male factor infertthty. Acknowledgement\: Grateful thanh\ I\ given to J. Bellrtnger and P Thornit\ for their a\\t\tance tn ti\\ue provision.
724 HYPERBARIC REPERFUSION
OXYGEN THERAPY EFFECT DAMAGE ON RAT TESTIS
Silvio, Siracusano Salvatore, Silvestre Gianmarco, Pecorari Valentina, Tiberio Anna. Belgrano Emanuele Stener
Department
IN
ISCHAEMIA-
d’Aloia
Gianluca,
of Urology. Univjersity of Trieste, Trieste. Italy
INTRODUCTION & OBJECTIVES: It is well known that the use of the hyperbaric oxygen significantly decreases the effects of the ischaemiareperfusion damage in animal model skin flaps. The aim of the present study is to evaluate the effects of the hyperbaric oxygen therapy on the trial model of the tat twisted and subsequently detwisted testis. MATERIALS & METHODS: We used 24 Wistar male rats, each weighting 300 g. and divided in 4 different groups (a-b-c-d) with a control rat for each group. All rats were subjected to a 720” torsion of the left testis, held in this position for 3 hours for groups A and C, and 6 hours for groups B and D. The subsequent removal of the left testis and of the healthy contralateral one, was petformed at 12,24 and 72 hours, 7 days and 14 days from the moment of the detwisting. In particular. groups C and D were submitted soon after the detwisting to an hyperbaric oxygen treatment for 60 minutes, using a 100% oxygen in an hyperbaric animal experimentation cylindrical watertight miniroom (model 2.50 NF, Sistemi Iperbarici Integrati), until a 2.8 A.T.A. pressure was reached. At last, the killed rats testis were caught and preserved in formol for the following macroscopic and microscopic evaluation according to these parameters: a) weight and size of the testis, b) mean tubular area, c) tubule/interstice ratio, d) edema (I to 5 score), e) congestion (1 to 5 score). f) germinal necrosis (1 to 5 score). g) Leydig cells (I to 5 score), h) epididymal necrosis ( I to 5 score). Afterwards. we made a comparative evaluation according to the aforesaid parameters. among the testis of each rat belonging to the same group and those ones of group A v5 group C, anti group B vs group D in relation with the hyperbaric treatment and ischaemia-reperfusion. The results were evaluated by univariate statistical analysis (one w’ay ANOVA) and by Wates corrected Chi Square Test. RESULTS: Thanks to the statistical analysis, we were able to see the inefficacy of the hyperbaric oxygen therapy in the ischeamia-reperfusion damage on the rat testis. European
Urology Supplements 1 (2002) No. 1, pp. 183
721 CAN VASOACTIVE INTESTINAL ISCHEMIAREPERFUSION INJURY RATS? Can’,
Tiire
F.‘,
Tunpel
N.*,
PEPTIDE OF TESTES
(VIP) PROTECT IN PREPUBERTAL
Uysal O.j, Gtirer F.‘, Ak D.?, Tunnel M.1
‘Department of Urology, Osmangazi University, Eskisehir, Turkey, ‘Department of Physiology, Osmangazi University. Eskisehir, Turkey, ‘Department of Histology-Embryology, Oamangazi University, Eskisehir, Turkey, Department of Analytical Chemistry, Anadolu University, Turkey INTRODUCTION & OBJECTIVES: Vasoactive intestinal peptide (VIP) is a member of a family of neuropeptides related to the hormone secretin. It is a bioactive peptide with broad biological effects on a widespread distribution in the body including testes. It has been reported that VIP protects many organs and the tissues against injury caused by oxidants and other toxins. In this study we investigated the protective effect of VIP on ischemia-reperfusion injury of the testes in prepubertal rata. MATERIALS & METHODS: Prepubertal (37-45 days old) male SpragueDawley rats were divided into 7 groups. Group I was the sham operated (n: 8). group II (n: 10) underwent testicular torsion for 2 h, groups III (n: 8) and V (n: IO) received saline vehicle and VIP respectively and underwent detortion for I h following 2 h of tortion. group IV (n: IO) and VI (n: 9) received saline vehicle and VIP respectively and underwent detortion for 4 h following 2 h of torsion. Group VII (n: 9) served to determine the basal values. Tortions were created by rotating the right testis 720” in a clockwise direction. Levels of auperoxide dismutase (SOD). catalase and malondialdehyde (MDA) were determined in testicular tissue. Histological evaluation was done in all groups. Difference was assessed using t-test and XL-test for parametric and nonparametric data respectively. RESULTS: In group II, the level of MDA was significantly increased when compared to the control groups (I and VII). The significant histologic tissue injury and mast cell degranulation were also observed between group II and control groups. In groups V and VI (VIP-treated), the levels of SOD and catalase were significantly decreased when compared to untreated groups (III. IV). Specimens from group III had a significantly greater histologic injury than group V. In groups III and IV, degranulation and exocytosis were observed in mast cells while most of the mast cells were granulated in groupe V and VI. The histochemical staining characteristics of mast cells were also different between VIP-treated and untreated groups. CONCLUSION:
VIP can protect testicular tissue from reperfusion
ATP: MORE THAN JUST ROCKET FUEL FOR SPERM? Thompson Cecil’, Mikhaihdis Dimitri’, Morgan Banks Frederick’, Calvert Robert’, Robert’, Burnstock Geoffrey’ ‘Urology, Royal Free Hospital. London, United Kingdom. ‘Chemical Pathology, Royal Free ZAutonomtc Neurowence Institute. Royal Free Hospital. London. United Kingdom, Hospital, London. United Kingdom
INTRODUCTION & OBJECTIVES:
Adenowte 5’.triphosphate (ATP) is known to be an tmportant energy source in sperm providmg fuel for motihty. In additton to its role in intrxellular energy metabolism, ATP i\ known to act as an extracellular rignalling molecule, affectmg the function and dtfferentiatton of various cell types via P2 purinoceptors (I). Testicular sperm are immotile. however motility is gained as they pass front the epididymal head to the tail. The mechanisms underlying this differentiation are unclear. ExpresGon of P2X purtnoceptor~ ha? been reported during various stages of rat \permatogenests. mdicative of a role during dtfferenttation (2). In additton, incubation of sperm with ATP has been found to stgnificantly improve in vitro fertilisation rates (3). The purpose of this study was to examine purinoceptor expression in epidtdymal sperm. and to examine any alteration during the maturation process leading to motility.
MATERIALS & METHODS: Testes were obtamed from euthanised mice, rats and hamsters Human tissue was obtained from appropriately consented patients undergoing orchidectomy for prostate or testicular cancer. Section\ were taken from both the head and tdil of the epididymi\, and placed adjacently on the same slide. lmmunohistochemistry was performed on the fixed sections using primary antibodies to each of the seven P2X receptor \ubtype\. RESULTS: P2XJ and P2X2 receptors were present on sperm contained wtthin the head of the epididymi\ from all species examined. This expression was undetectable on sperm in the epidtdymal tall m all species except for humans tn which xmte expression persisted. P2X3 exprewon was observed on epididymal head, but not tail sperm, from the mouse and the hamster only. P2X4 expression was observed in the mouse and human sperm from both the head and tail of the epidtdymir. P?XS expression was observed to a similar degree in both the head and the tad eptdtdymal sperm from the tnouse. P2X6 and P2X7 staining was not observed in sperm from etther the head or tail of the epidtdymis from any species. In control experiment\, no immunoreactivity was observed when the primary antibody was omttted and was pre-abhorbed. CONCLUSION: Thih study uniquely demonstrates that beveral P2X purmoceptors subtype\ are expressed on epididymal sperm, although there are some species differences. There is strong expression on immature sperm contained within the head of the epididymis, and a wbxquent. but variable loss of expression on more mature sperm contained within the caudal eptdidymis. Sperm undergo significant maturation in the epididymis. Altered sperm expression of purinergic receptorc may be involved in this process. ATP has already been shown to nnprove fertility in vttro (3). Specific targeting of these receptors may lead to therapeutic agents. vvhich might be effective in v’ivo. Reference?, al Cell Tis\
injury.
I. Abbracchio M.P., Burnstock G. Jpn J Pharmacol 358, 139.145 2. Glass R. et Org 2001: 16Y: 377-3X7 3. Ro\sato M. et al Hum Repr lY99:14 (3) 694.hY7.
723 ULTRASTRUCTURE AND PHARMACOLOGY TESTICULAR CAPSULE CONTRACTION Banks Frederick’, Burnstock Geoffrey’ ‘Urology. Ho\pttal, Hospital,
Calvert
Robert’.
Turmaine
Mark’.
Knight
OF THE Gill’.
Morgan
HUMAN Robert’.
Royal Free Ho\pttal, London, United Kingdom, ‘Anatomy. UmverGty College ‘Autonomic Neumxiencc In\tttute. Royal Free London, United Kingdom, London. United Kingdom
INTRODUCTION & OBJECTIVES: Sperm contained
within the tests\ are immotile. The mechanism by which aperm move front the testis to the epidtdymt? i\ poorly understood. In thi\ study we demonstrate smooth muscle wtthin the human testicular capsule (tunica albuginea). and charactertse tts contractile response. Contraction of the testicular capwlc may ha\e a de in expelling sperm out of the testis into the epidtdymw
MATERIALS & METHODS: Following prior ethical approval and Informed congent. testicular capsular tissue wa\ taken from patients undergoing orchidectomy for either pro\tattc or testicular cancer, or for gender reassignment (n=S). Tiswe for pharmacological evaluation was immediately w\pended m organ baths containing oxygenated Kreb’s solution at 3&“C. Tt~we was pretensioned to Ig and subJected to electrical field \ttmulation (EFS) of autonomic newes at I00 v. 0.3 m\, 30 s ev’ery 5 min. in the frequency range 0.5-32 HI. Following this. exogenom noradrenalme (NA). (single dohe\ IO nmol-0 3 mmol) acctylcholine (ACh) (cumulative doses IO nmol-0.3 mmol) and adeno\ine 5‘ triphosphate (ATP) (single do\e\ nm-0.3 mmol) were added to e\tabh\h conccntrationrrpon\e curve\. Tiwte wxtion\ were examined ustng transmtssion clcctrott microscopy. Further sectton\ underwent immunohi\tological evaluation using c\tabli\hed anti smooth muscle myoatn anttbodiej
IO0
RESULTS: Concentratton dependent contraction was observed to NA. giving an EC50 equivalent to I .hS b,mol. Rcsponaea to nerve ~timulatmn were small and inconsistent and thaw to cxogenoualy applied ACh and ATP were meffective. Immunohi\tological studieh demonstrated small clusters of smooth tmuscle cells throughout the capsule. No obvious pattern or layer formation of smooth muscle was obwwd itt the capsule. Electron micrwcopy demonstrated smooth muxle cell\. apparently not in any eabdy recogniwble at-rangement. although they vvere for the mo\t pnrt arranged longitudmally. Interconnecting proce\\es and gap ,junctions between muscle cells vvere xxm. The neural component wa\ sparse, although. characteristic neuronal vanco\ttte\ containing synaptic v’esicleh vvere observed in tmmedtate proxtmity to smooth muwle cell\. CONCLUSION: Smooth muxlc cell\ are present wtthtn the human testicular capsule. The\e cell\ are re\pon\ivc to NA in a concentration~dependent manner. This suggests that te\ttctdx capsular contractton may be under lympathetic nerv’ou\ system control. Testicular contraction may have a physiological role tn expelling sperm out 01 the testis into the cpididymi\. Stimulation of such contractiott may be relevant in the treatment of male factor infertthty. Acknowledgement\: Grateful thanh\ I\ given to J. Bellrtnger and P Thornit\ for their a\\t\tance tn ti\\ue provision.
724 HYPERBARIC REPERFUSION
OXYGEN THERAPY EFFECT DAMAGE ON RAT TESTIS
Silvio, Siracusano Salvatore, Silvestre Gianmarco, Pecorari Valentina, Tiberio Anna. Belgrano Emanuele Stener
Department
IN
ISCHAEMIA-
d’Aloia
Gianluca,
of Urology. Univjersity of Trieste, Trieste. Italy
INTRODUCTION & OBJECTIVES: It is well known that the use of the hyperbaric oxygen significantly decreases the effects of the ischaemiareperfusion damage in animal model skin flaps. The aim of the present study is to evaluate the effects of the hyperbaric oxygen therapy on the trial model of the tat twisted and subsequently detwisted testis. MATERIALS & METHODS: We used 24 Wistar male rats, each weighting 300 g. and divided in 4 different groups (a-b-c-d) with a control rat for each group. All rats were subjected to a 720” torsion of the left testis, held in this position for 3 hours for groups A and C, and 6 hours for groups B and D. The subsequent removal of the left testis and of the healthy contralateral one, was petformed at 12,24 and 72 hours, 7 days and 14 days from the moment of the detwisting. In particular. groups C and D were submitted soon after the detwisting to an hyperbaric oxygen treatment for 60 minutes, using a 100% oxygen in an hyperbaric animal experimentation cylindrical watertight miniroom (model 2.50 NF, Sistemi Iperbarici Integrati), until a 2.8 A.T.A. pressure was reached. At last, the killed rats testis were caught and preserved in formol for the following macroscopic and microscopic evaluation according to these parameters: a) weight and size of the testis, b) mean tubular area, c) tubule/interstice ratio, d) edema (I to 5 score), e) congestion (1 to 5 score). f) germinal necrosis (1 to 5 score). g) Leydig cells (I to 5 score), h) epididymal necrosis ( I to 5 score). Afterwards. we made a comparative evaluation according to the aforesaid parameters. among the testis of each rat belonging to the same group and those ones of group A v5 group C, anti group B vs group D in relation with the hyperbaric treatment and ischaemia-reperfusion. The results were evaluated by univariate statistical analysis (one w’ay ANOVA) and by Wates corrected Chi Square Test. RESULTS: Thanks to the statistical analysis, we were able to see the inefficacy of the hyperbaric oxygen therapy in the ischeamia-reperfusion damage on the rat testis. European
Urology Supplements 1 (2002) No. 1, pp. 183