Overexpression of endothelial nitric oxide synthase in transgenic mice accelerates testicular germ cell apoptosis induced by experimental cryptorchidism.

Surgical induction of cryptorchidism in experimental animals causes testicular germ cell apoptosis and infertility. The mechanisms of germ cell apoptosis have been associated with oxidative stress or testicular exposure to elevated temperature. Nitric oxide (NO) has been associated with apoptosis in a number of cell types. The objective of this study was to investigate whether overexpression of endothelial NO synthase (eNOS) could accelerate apoptosis of germ cells in the testes of transgenic mice. There are 3 NOS isoforms, and we restricted the analysis to eNOS at this time. For the colocalization of eNOS, staining in degenerating germ cells that were apoptotic cells suggested that eNOS may be related to germ cell apoptosis. eNOS overexpression in the testes of eNOS transgenic (eNOS-Tg) mice was examined using Western blot analysis. Unilateral cryptorchidism was surgically induced in both eNOS-Tg and wild-type (WT) adult mice. The testes were evaluated 1, 3, 5, 7, and 14 days after the operation by weighing the testes and examining histopathologic features and cell apoptosis using in situ microscopic analysis of DNA fragmentation. Immunoblotting for eNOS protein demonstrated increases in eNOS protein expression in testes, as well as the lung and aorta. In eNOS-Tg mice, weight reduction of cryptorchid testis was significantly increased on days 3, 5, and 7 (P = .02, .02, and .04, respectively). The numbers of spermatocytes and spermatids of eNOS-Tg cryptorchid testis significantly decreased compared with those of WT cryptorchid testis from day 3 (spermatocytes: P = .04; spermatids: P = .02). Moreover, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling demonstrated that eNOS-Tg mice significantly accelerate germ cell apoptotic changes induced by experimental cryptorchidism compared with WT mice from day 3 (P = .03). We have provided evidence that eNOS plays a functional role in mouse spermatogenesis in cryptorchidism-induced apoptosis.

Recombinant interleukin-2 enhanced the antitumor effect of ADV/RSV-HSV-tk/ACV therapy in a murine bladder cancer model.

BACKGROUND: Previous studies demonstrated the antitumor effects of IL-2 and ADV/RSV-HSV-tk in bladder tumor models. In our study, we employed the intramuscular injection of recombinant IL-2 combined with ADV/RSV-HSV-tk gene therapy in the MBT-2 murine bladder tumor model. MATERIALS AND METHODS: In the in vitro study, after adenoviral gene transduction efficiency had been assessed, the cytotoxicity of ADV/RSV-HSV-tk/ACV was examined. In the in vivo study, ADV/RSV-HSV-tk was injected into MBT-2 subcutaneous tumors, ACV was injected intraperitoneally daily for 13 days and recombinant IL-2 was injected intramuscularly daily for 10 days. RESULTS: The X-gal staining of MBT-2 cells infected with 125 multiplicity of injection (MOI) indicated > 20% adenoviral gene transduction efficiency. The cell growth of MBT-2 infected with 125 MOI was significantly inhibited by 40 microM of ACV. In the in vivo study, the combination therapy significantly inhibited tumor growth in the MBT-2 tumor model. CONCLUSION: The systemic administration of recombinant IL-2 in combination with HSV-tk gene therapy exhibited an enhanced antitumor effect.

Erythropoietin gene transfer into rat testes by in vivo electropo-ration may reduce the risk of germ cell loss caused by cryptorchidism.

AIM: To investigate the effects of rat Erythropoietin (Epo) on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. METHODS: Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups: the first group was given intratesticular injections of pCAGGS-Epo (pCAGGS-Epo group), the second group was given intratesticular injections of pCAGGS (pCAGGS group), and the third group were given intratesticular injections of phosphate-buffered saline (PBS group). At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells (G/S ratio) was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. RESULTS: The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was (0.85+/-0.08) g and (0.83+/-0.03) g, respectively, at week 1 (P = 0.788) and (0.62+/-0.06) g and (0.52+/-0.02) g, respectively, at week 2 (P = 0.047). At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 +/-6.80 vs. 18.63+/-5.30 at week 1 (P = 0.0078) and 7.16+/-3.06 vs. 6.05+/-1.58 at week 2 (P = 0.1471), respectively. CONCLUSION: The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.

Serum testosterone levels in patients with nonmosaic Klinefelter syndrome after testicular sperm extraction for intracytoplasmic sperm injection.

We measured testosterone levels in 24 patients with nonmosaic Klinefelter syndrome before and at 6 and 12 months after conventional or microdissection testicular sperm extraction. Testosterone levels decreased after surgery by either technique, and they did not recover to baseline concentrations, even when using less invasive microdissection techniques.

Pregnancy by insemination of cryopreserved spermatozoa from a man with retrograde ejaculation: a case report.

BACKGROUND: Owing to the prevalence of diabetes mellitus, spinal injuries and aggressive surgical treatment of cancer, the number of younger patients with retrograde ejaculation is increasing. Since medical treatment to restore antegrade ejaculation often fails, several options for accomplishing insemination by these patients, including the use of sperm-rich urine obtained after masturbation and in vitro fertilization with sperm retrieved from the seminal tract, have been reported. We used the least invasive and most inexpensive procedure in a patient/couple with this condition. CASE: A 23-year-old man suffered from retrograde ejaculation after a spinal injury. He could achieve erection and engage in sexual intercourse but seldom had an orgasm or the sensation of ejaculation. We obtained spermatozoa from urine produced after masturbation at home and froze them. We used these frozen-thawed spermatozoa for intrauterine insemination, leading to the term birth of a healthy infant. CONCLUSION: In selected patient/couples, frozen spermatozoa obtained from postmasturbation urine can be used successfully for intrauterine insemination. This minimally invasive and most inexpensive procedure should be tried before planning in vitro fertilization.

Midkine promoter-based conditionally replicative adenovirus for targeting midkine-expressing human bladder cancer model.

OBJECTIVES: To develop a novel therapeutic strategy against human bladder cancer using Ad-MK-E1a-a midkine (MK) promoter-regulated, conditionally replicating, adenovirus. METHODS: We tested several human cancer cell lines in vitro, including those of bladder cancer (KK47, 5637, and T24), lung cancer (A549), and head and neck cancer (H891). In each cell line, we examined MK mRNA expression by TaqMan real-time quantitative polymerase chain reaction, MK promoter activity, after plasmid transfection, using a luciferase assay, and the transduction efficiency by co-transfection with the cytomegalovirus-beta-gal plasmid. In these cells, we assessed the cell type-specific replication of Ad-MK-E1a virus by measuring the E1a DNA copy number by real-time polymerase chain reaction and the cell growth inhibition due to this virus using the Alamar blue assay. In animal studies, nude mice were subcutaneously inoculated with KK47 cells and later intratumorally injected with phosphate-buffered saline or Ad5-CMV-LacZ or Ad-MK-E1a. RESULTS: The MK mRNA expression level and MK promoter-driven luciferase activity were relatively greater and markedly increased, respectively, in the 5637, A549, and KK47 cells than in the T24 and H891 cells. After Ad-MK-E1a infection, the E1a DNA copy number increased more significantly in the KK47, 5637, and A549 cells than in the T24 and H891 cells. At a multiplicity of infection of 0.01, Ad-MK-E1a significantly inhibited KK47 and 5637 cell growth. In vivo, Ad-MK-E1a injection markedly inhibited KK47 tumor growth. CONCLUSIONS: We have demonstrated the antitumor effect of Ad-MK-E1a in a human bladder cancer model overexpressing MK mRNA.

Progress report on phase I/II clinical trial of Ad-OC-TK plus VAL therapy for metastatic or locally recurrent prostate cancer: Initial experience at Kobe University.

There is no effective therapy for hormone-refractory prostate cancer and a novel therapeutic modality, such as a gene therapy, should be actively pursued. Previously, Gardner and Chung conducted a phase I clinical trial of Ad-OC-TK (recombinant adenoviral vector containing osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) for the treatment of hormone-refractory prostate cancer at the University of Virginia. We report on our ongoing phase I/II clinical trial of Ad-OC-TK plus VAL for the treatment of advanced prostate cancer at the Kobe University Hospital, Japan.

Four pregnancies in nonmosaic Klinefelter's syndrome using cryopreserved-thawed testicular spermatozoa.

OBJECTIVE: To investigate feasibility of using cryopreserved-thawed testicular spermatozoa from patients with nonmosaic Klinefelter's syndrome for intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: University-based hospital and IVF clinic. PATIENT(S): Six patients with nonmosaic Klinefelter's syndrome who underwent testicular sperm extraction for ICSI. INTERVENTION(S): Microdissection testicular sperm extraction (TESE) and ICSI. MAIN OUTCOME MEASURE(S): We compared results of ICSI using cryopreserved testicular spermatozoa with those previously reported in Klinefelter's syndrome and those in nonobstructive azoospermia patients using cryopreserved testicular spermatozoa at our institution with respect to embryo cleavage rate, implantation rate, and pregnancy outcome. RESULT(S): Four of six patient couples with successful microdissection TESE achieved pregnancy using cryopreserved-thawed testicular spermatozoa. One pregnancy resulted in early-pregnancy abortion, two in delivery of healthy singleton girls, and one delivery of a healthy singleton boy. CONCLUSION(S): Cryopreserved-thawed testicular spermatozoa can be used successfully for ICSI in patients with nonmosaic Klinefelter's syndrome.

Age as a limiting factor for successful sperm retrieval in patients with nonmosaic Klinefelter's syndrome.

OBJECTIVE: To determine factors affecting successful sperm retrieval by testicular sperm extraction in patients with nonmosaic Klinefelter's syndrome. DESIGN: Medical record analysis for nonmosaic Klinefelter's syndrome patients who underwent testicular sperm extraction. SETTING: Three university-based tertiary centers. PATIENT(S): Fifty-one patients with nonobstructive azoospermia related to nonmosaic Klinefelter's syndrome. INTERVENTION(S): Testicular sperm extraction. MAIN OUTCOME MEASURE(S): Correlation of patient characteristics; serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T); as well as testicular volume with success in testicular sperm extraction. RESULT(S): We succeeded in obtaining spermatozoa in 26 patients and failed in 25. Levels of LH, FSH, and T and testicular volume did not differ between patient groups defined by success and failure. Median ages for successful and failed testicular sperm extraction were 31 and 38 years, respectively (statistically significant difference). When we analyzed success rates of testicular sperm extraction between two patient groups (<35 and > or =35 years old), the percentage of successful recovery of spermatozoa decreased after the age of 35 years (statistically significant difference). CONCLUSION(S): Testicular sperm extraction should be recommended before the critical age of 35 years.

Cut-to-the-light technique and potassium titanyl phosphate laser ureterotomy for complete ureteral obstruction.

We describe a case of complete ureteral obstruction managed by endoscopic recanalization using a 'cut-to-the-light' technique followed by potassium titanyl phosphate (KTP) laser ureterotomy. A 53-year-old man developed a ureteral obstruction following the transurethral resection of a bladder tumor (TUR-Bt) at the left ureteral orifice. The length of the obstructed segment was estimated at 1 cm based on combined antegrade and retrograde contrast studies. Histopathological analysis indicated that the obstruction was caused by fibrosis. The 'cut-to-the-light' technique was used for recanalization, and KTP laser ureterotomy was performed to obtain an adequate ureteral lumen. A 14 F/7 F endoureterotomy stent was removed 6 weeks after the operation. No significant complications and no signs of stenosis were observed 24 months after endoscopic repair. Endoscopic recanalization is a safe, effective technique for the management of a completely obliterated ureteral segment, especially in combination with subsequent KTP laser ureterotomy.

Adenoviral-mediated HGF expression inhibits germ cell apoptosis in rats with cryptorchidism.

BACKGROUND: Prior studies have shown that the hepatocyte growth factor (HGF), as known for its multiple biological effects, possibly regulates spermatogenesis or tubulogenesis in the testis. To clarify the effect of HGF on restoration of spermatogenesis, or testicular weight, we transferred the HGF gene into the testis of the rat experimental cryptorchid model. METHODS: Replication-deficient recombinant adenoviral vectors containing the CAG promoter driving rat HGF (pAxCAHGF) and LacZ (pAxCALacZ) were constructed. Sprague-Dawley rats surgically induced with unilateral cryptorchidism and subsequent orchidopexy were divided into three groups: control (PBS), pAxCALacZ and pAxCAHGF by intratesticular injection. At 2 and 4 weeks after subsequent orchidopexy, testes were removed and weighed. These specimens were analyzed histopathologically, and examined for cell apoptosis. HGF expression in these specimens associated with c-Met receptor-mediated signal molecules was examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot or immunohistochemical study. RESULTS: Adenovirus-mediated HGF gene transfer induced overexpression of HGF in some seminiferous epithelial cells and interstitial cells, increased the phosphorylation of ERK and Akt, and decreased numbers of apoptotic cells of germ cells. HGF transduction also significantly increased the numbers of germ cells and testicular weight by 4 weeks compared with the other control groups. CONCLUSIONS: Adenoviral-mediated HGF gene transfer into the testis in the cryptorchidism rats inhibited germ cell apoptosis and restored spermatogenesis.