Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009 THE JOURNAL OF UROLOGY® 685

1896A NEW HYPOTHESIS ON VARICOCELE PHYSIOPATHOLOGY: THE VASODILATION EFFECTS OF TESTOSTERONE ON HUMAN ISOLATED INTERNAL SPERMATIC VEINS VIA ATP-SENSITIVE POTASIUM CHANNELS

Melik Seyrek*, Hasan Cem Irkilata, Ismail M Vural, Ibrahim Yildirim, Seref Basal, Oguzhan Yildiz, Murat Dayanc, Ankara, Turkey

INTRODUCTION AND OBJECTIVES: In our previous study, we demonstrated that testosterone induces relaxation responses by a dose dependent manner in human isolated internal spermatic veins and the vasodilatory effect of testosterone decreases in high grade varicoceles. But the mechanism of the testosterone-induced relaxant responses is not exactly known. In this study we aimed to investigate the possible mechanism of testosterones vascular action.

METHODS: Responses of isolated internal spermatic veins were recorded isometricly by a force displacement transducer. After contracting venous strips with 48 mM KCl control relaxations of testosterone (0.1-300 μl/ml) were recorded. Following the washing period, the rings were incubated with non-selective large conductance calcium activated potasium (K+) channel (BKCa) and voltage-dependent K+ channel (KV) inhibitor tetraethylammonium (TEA, 1 mM), ATP-sensitive K+ channel (KATP) inhibitor glibenclamide (GLI, 10 μM) or KV inhibitor 4-aminopyridine (4-AP) for 30 min; and after contracting the strips with 48 mM KCl relaxations to testosterone (0.1-300 μl/ml) were repeated.

RESULTS: Testosterone induced relaxation responses in human isolated internal spermatic veins (Emax:42.10,n=12). Highest concentration of the vehicle (1.5% ethanol) had no significant relaxant effect. While TEA and 4-AP did not alter testosterone-induced relaxant responses, GLI inhibited these responses.

CONCLUSIONS: These results demonstrated that testosterone induced relaxation responses in human isolated internal spermatic veins via ATP-sensitive K+ channels.

Source of Funding: None

1897SPERM APOPTOSIS IS ACTIVATED THROUGH THE MITOCHONDRIAL PATHWAY

Howard H Kim*, Marc Goldstein, Darius A Paduch, New York, NY

INTRODUCTION AND OBJECTIVES: In the future, healthy sperm may be selected by flow cytometry (FC) for use in ICSI. Apoptosis is associated with sperm chromatin damage. Although FC analysis of apoptosis has been reported for many cell types, these assays have not been completely characterized for sperm. Sperm have key differences from other cells including reduced cytoplasmic volume. Apoptosis is dependent on activation of mitochondrial and cytoplasmic enzymes. We tested the mechanisms of apoptosis activation and chromatin damage in sperm using markers for 2 distinct stages of apoptosis.

METHODS: Semen samples of 12 men were used to optimize assay conditions for the fluorescent dyes Annexin V (A-V) and DiIC1(5) and analyzed with FC. A-V binds to phosphatidylserine (PS) on the inner side of cell membranes during apoptosis. DiIC1(5) is a marker for polarized mitochondrial membrane potential in healthy cells and has a reduced signal in apoptotic cells with depolarized mitochondrial membranes. Apoptosis was induced in half of each sample by incubation with sodium nitroprusside. The staining of the sperm before and after induction (of apoptosis) were compared. Statistical significance was determined by the chi-square test.

RESULTS: In sperm before induction, 2.8% were A-V(+) and DiIC1(5)(+) and 33.5% were A-V(+) and DiIC1(5)(-). After induction, 4.3% were A-V(+) and DiIC1(5) (+) and 41.4% were A-V(+) and DiIC1(5)(-). A-V signal increased and DiIC1(5) signal decreased significantly after induction (Figure 1a). DiIC1(5) and A-V are effective markers of apoptosis in sperm. We elucidated the time course of early apoptosis in sperm by demonstrating the crescendo of PS binding by A-V and the tapering of disrupted mitochondrial membrane potential (reduced DiIC1(5) signal) 2 hours after induction. The magnitude of signal change increased (9.4% to

13.3%) for A-V and decreased (4.4% to 1.0%) for DiIC1(5) (Figure 1b). CONCLUSIONS: Sperm chromatin damage is a result of

apoptosis induction through mitochondrial, not cytoplasmic pathways, which confirms intuitive assumptions, as sperm have low cytoplasmic volume. The time response curve indicates that changes in mitochondrial potential precede the changes in membrane phospholipids.

Source of Funding: The Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust and The Ferdinand C. Valentine Fellowship of the New York Academy of Medicine

1898A LIGHT AND ELECTRON MICROSCOPY STUDY OF THE EFFECTS OF EXPERIMENTAL UNILATERAL CRYPTORCHIDISM ON THE SPERMATOGENESIS OF THE CONTRALATERAL TESTIS IN ADULT RABBITS

Fotios Dimitriadis*, Yonago, Japan; Eleni Kaldrymidou, Dimitra Psalla, Thessaloniki, Greece; Panagiota Tsounapi, Takeshi Watanabe, Yonago, Japan; Nikolaos Sofikitis, Ioannnina, Greece; Ikuo Miyagawa, Yonago, Japan

INTRODUCTION AND OBJECTIVES: Despite general agreement that cryptorchidism leads to spermatogenic damage of the abdominal testis, the extent of the damage to the contralateral spermatogenic ability remains controversial. We examined the effects of experimental unilateral cryptorchidism on the population of germ cells and other ultrastructural characteristics of the contralateral testis in adult rabbits.

METHODS: Right unilateral cryptorchidism was induced in 20 mature male New Zealand white rabbits (group A) by returning the testis to the abdominal cavity via a surgical procedure. Another group of 20 age-matched control animals underwent sham operation (group B). Seventeen weeks after the operation the fertilization ability of group A and B has been evaluated. Ten rabbits from each group have been individually placed together with one female rabbit of proven fertility for 24 hours. Serum testosterone levels before and after administration of 5000U human chorionic gonadotropin (hCG) has been assessed as well. Thereafter, both abdominal and scrotal testes were removed from all animals. Number and morphology of germ cells, seminiferous tubules diameter and area, and ultrastructural details of the testes have been estimated with both light and transmission electron microscopy using paraffin or epoxy resin embedded sections respectively.

RESULTS: Cryptorchidism resulted in severe testicular atrophy and spermatogenic arrest in the abdominal testis. The seminiferous epithelium was composed of only spermatogonia type A and Sertoli cells. The number of type A spermatogonia per testis was reduced by 82%, whereas the diameter and area of the seminiferous tubules were decreased by 47,45% and 72,17% respectively. The contralateral testis demonstrated significant changes as well, including thickening of the basement membrane, reduction in germ cell lines, vacuolization, margination of chromatin and increased volumes of Sertoli cell lipid droplets (P<0.05). The fertilization rate was significantly lower in group A than in group B (P<0.05). The serum base testosterone levels in group A were within the normal values but in lower levels in comparison with group B (P<0.05). The testosterone response in hCG was significantly smaller in group A than in group B (P<0.05).

CONCLUSIONS: Unilateral cryptorchidism caused substantial adverse effects to the contralateral spermatogenic ability which may explain the decrease in fertility in rabbits with unilateral undescended testis.

Source of Funding: None