Noritoshi Enatsu

Efficient Transfection of DNA into Primarily Cultured Rat Sertoli Cells by Electroporation

ABSTRACT The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P < 0.001). Cell survival clearly decreased following one to three pulses, from 83.9% ± 6.1% to 3.2% ± 1.1%, with EGFP expression increasing from 41.8% ± 9.4% to 66.7% ± 5.2%. The yield of positive cells increased with increasing concentration of plasmid DNA (range, 10–50 μg/ml), from 14.0% ± 2.8% to 35.0% ± 6.3%, but cell viability steadily decreased following 20 μg/ml plasmid DNA, from 73.1% ± 4.9% to 57.0% ± 6.6%. Compared with two popular cationic lipid transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P < 0.001). We describe the process of optimizing electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicate that the method of electroporation is more suitable than other approaches for the transfection of Sertoli cells.

Assessment of Time-dependent Changes in Semen Parameters in Infertile Men After Microsurgical Varicocelectomy

OBJECTIVE To characterize the changes in seminogram findings in infertile men after varicocelectomy. METHODS This study included 71 consecutive infertile men who underwent microsurgical low ligation varicocelectomy and received 3 semen analyses, 1 before microsurgical varicocelectomy and again at 3 and 12 months after. Total motile sperm count (TMSC) was calculated using the following formula: [volume (mL) × concentration (millions/mL) × motility (%)]. RESULTS Despite the lack of significant changes in the proportion of sperm with abnormal morphology, sperm concentration, motility, and TMSC in the 71 patients were significantly higher at 3 and 12 months after varicocelectomy than before surgery. However, no further improvement in these parameters at 12 months after varicocelectomy was noted compared with those at 3 months. Furthermore, when the included men were divided into 3 groups according to preoperative TMSC as <3 million, 3-9 million, and >9 million, TMSCs at 3 months after varicocelectomy in all 3 groups were significantly higher than those before varicocelectomy; however, TMSCs at 12 months after surgery in all groups were similar to those at 3 months. CONCLUSION The level of improvement in semen parameters at 3 months after varicocelectomy may be stable at 12 months after surgery, irrespective of baseline values of TMSC. Therefore, varicocelectomy could be offered as a therapeutic option for infertile men, even for couples with an older woman, because its efficacy is evaluable at 3 months after surgery, and assisted reproductive technology could be immediately applied to ineffective cases.

Management of non-obstructive azoospermia

Non-obstructive azoospermia (NOA) is defined as no sperm in the ejaculate due to failure of spermatogenesis and is the most severe form of male infertility. The etiology of NOA is either intrinsic testicular impairment or inadequate gonadotropin production. Chromosomal or genetic abnormalities should be evaluated because there is a relatively high incidence compared with the normal population. Although rare, NOA due to inadequate gonadotropin production is a condition in which fertility can be improved by medical treatment. In contrast, there is no treatment that can restore spermatogenesis in the majority of NOA patients. Consequently, testicular extraction of sperm under an operating microscope (micro-TESE) has been the first-line treatment for these patients. Other treatment options include varicocelectomy for NOA patients with a palpable varicocele and orchidopexy if undescended testes are diagnosed after adulthood, although management of these patients remains controversial. Advances in retrieving spermatozoa more efficiently by micro-TESE have been made during the past decade. In addition, recent advances in biotechnology have raised the possibility of using germ cells produced from stem cells in the future. This review presents current knowledge about the etiology, diagnosis, and treatment of NOA.

Improved Lower Urinary Tract Symptoms Associated With Testosterone Replacement Therapy in Japanese Men With Late-Onset Hypogonadism

This study aimed to investigate the effects of testosterone replacement therapy (TRT) on lower urinary tract symptoms (LUTS) in men with late-onset hypogonadism (LOH) and to identify parameters predicting the efficacy of TRT in improving LUTS. This study included 60 consecutive Japanese men who were diagnosed with LOH and subsequently received TRT between January 2009 and December 2014. In this series, 250 mg of testosterone was injected intramuscularly every 3 or 4 weeks in all patients. The following parameters were retrospectively reviewed: body mass index (BMI), Aging Male Symptom (AMS) score, International Prostate Symptom Score (IPSS), International Index of Erectile Function–5 (IIEF-5) score, residual urine volume, prostate volume, serum levels of the prostate-specific antigen (PSA), and total- and free-testosterone levels before and 6 months after TRT. No significant differences were observed in BMI, residual urine volume, or prostate volume between surveys before and after TRT. The AMS score, IPSS, and IIEF-5 score were significantly improved and significant increases were noted in the serum levels of PSA and total- and free-testosterone levels after TRT. An analysis of IPSS subscores documented the significant improvement in storage symptom scores, but not in voiding symptom scores after TRT. Multivariate analyses of parameters assessed in this study identified the pretreatment AMS score, posttreatment IIEF-5 score, and prostate volume as independent predictors of improvements in IPSS following TRT. This study revealed that TRT appeared to have considerable therapeutic effects on LUTS, particularly on storage symptoms, in men with LOH.

Erectile function and its impact on quality of life in Japanese men on hemodialysis

Abstract The objective of this study was to characterize the erectile function and its impact on health-related quality of life (HRQOL) in Japanese men on hemodialysis. This study included 65 consecutive Japanese men <80 years on hemodialysis. Erectile function and HRQOL were evaluated using the Sexual Health Inventory for Men (SHIM) and the Short-Form 8 (SF-8) survey, respectively. These 65 men were classified into 54 with SHIM ≤11; relatively severe erectile dysfunction (ED) (group A) and 11 with SHIM ≥12; relatively mild ED (group B). There were significant differences in age, marital status, ankle-brachial index (ABI), and serum levels of free testosterone and prolactin between groups A and B. Of several factors examined, age, marital status and ABI were significantly associated with the severity of ED on univariate analysis, and of these 3 factors, only age was significantly associated with severity of ED on multivariate analysis. SF-8 survey revealed that all scale scores in group A were inferior to those in group B. Comparatively severe ED appeared to be frequently observed in Japanese men on hemodialysis, and this trend was marked in elderly men. Furthermore, the severity of ED had a negative impact on the wide range of HRQOL in these men.

Prospective assessment of health-related quality of life in men with late-onset hypogonadism who received testosterone replacement therapy

The objective of this study was to characterise the status of health‐related quality of life (HRQOL) in Japanese men with late‐onset hypogonadism (LOH) treated with testosterone replacement therapy (TRT). HRQOL in 69 consecutive Japanese men with LOH undergoing TRT for at least 6 months was prospectively evaluated before and 6 months after the initiation of TRT using the Medical Outcomes Study 8‐Item Short‐Form Health Survey (SF‐8). All eight‐scale scores except for bodily pain (BP) in the 69 patients at 6 months after the introduction of TRT significantly improved compared with those before TRT; however, all scale scores except for BP in the 69 patients were significantly inferior to those in age‐matched Japanese controls irrespective of the timing of SF‐8. Multivariate analyses of several parameters revealed that both age and Aging Male Symptom (AMS) score had an independent impact on mental health (MH), despite the lack of an independent association between any score and the remaining factors examined. TRT appeared to significantly improve the status of HRQOL in men with LOH; however, even after the introduction of TRT, HRQOL associated with MH remained significantly impaired in elderly men and/or those with a high AMS score.

Effects of dutasteride on serum free-testosterone and clinical significance of testosterone changes

Sixty‐two patients with benign prostate hyperplasia (BPH) who were being treated with dutasteride participated in this study. Prostate volume, uroflowmetry, blood tests, the International Prostate Symptom Score (IPSS) and International Index of Erectile Function (IIEF‐5) were determined before and 1, 3 and 12 months after the treatment with dutasteride. Patients were divided into two groups based on changes in serum testosterone after 1 month: Group A (>20% increase; n = 33) or Group B (<20% increase; n = 29). Serum free‐testosterone levels were 20.4% higher after 1 month and remained constant thereafter. When Groups A and B were compared, baseline free‐testosterone levels were significantly lower in Group A, IPSS QOL was significantly better in Group A at 3 and 12 months, and no significant differences were observed in uroflowmetry, prostate volume, IPSS or IIEF‐5. A univariate analysis identified serum free‐testosterone levels and the IPSS storage symptom subscore as significant factors influencing IPSS QOL at 12 months, and only the IPSS storage symptom subscore appeared to be independently related to IPSS QOL. These results indicate that dutasteride increases serum free‐testosterone levels in BPH patients, particularly with low baseline free‐testosterone levels, and the increase in free‐testosterone may have further add‐on impacts on their urinary tract symptoms.

Identification of Spermatogenically Active Regions in Rat Testes by Using Narrow-band Imaging System

OBJECTIVE To evaluate the feasibility of narrow-band imaging (NBI) system in microdissection testicular sperm extraction (micro-TESE). MATERIALS AND METHODS Firstly, we investigated angiogenic potential in human testicular specimens obtained from 48 patients who underwent micro-TESE. We then created a testicular injury model in rats with a single topical injection of cisplatin into the testes, and the testes were observed with and without NBI. To assess the relation between pathological changes and visual images, an immunofluorescence study of blood vessels in rat testes was carried out. We finally conducted an experiment that assumed micro-TESE by using a nonobstructive azoospermia (NOA) rat model induced by busulfan. RESULTS The number of blood vessels in the specimens of hypospermatogenesis was significantly greater than that in the specimens of maturation arrest and Sertoli cell only. In rat testes, cisplatin caused atrophic changes, and significant differences in visual color between atrophic and normal lesions were noted using NBI. The number of seminiferous tubules with spermatozoa in atrophic regions was significantly lower than that in normal regions, and a significantly small number of blood vessels in atrophic regions was also noted compared with that in normal regions. NBI also identified patchy spermatogenesis in the busulfan-induced NOA rat model. CONCLUSION An NBI system can distinguish spermatogenically active regions through the visualization of blood vessels in rat testes. This system may have the potential to provide useful information during micro-TESE for men with NOA.

Dutasteride-mediated morphological changes in the genitourinary tract associated with altered expression patterns of the androgen and estrogen receptors in male rats

We evaluated the effects of dutasteride on the genitourinary tract using fifteen 8‐week‐old male Sprague–Dawley rats. Animals were divided into three groups comprising five animals each and treated as follows. Group A was a control group, members of Group B received oral administration of dutasteride 0.1 mg/kg/day from the age of 8 to 16 weeks, and members of Group C were castrated at the age of 8 weeks. All rats were killed at the age of 16 weeks for the sample collection of blood, bladder, prostate, seminal vesicles, and penis. Then, we evaluated the pathological examination for evaluating the tissue fibrosis and hormonal receptor expression. The results showed that the mean size of the prostate and seminal vesicles was smaller in Group B and Group C than in Group A. Serum and tissue concentrations of both testosterone and dihydrotestosterone were remarkably reduced in serum and all tissues in Group C compared with Group A. On the other hand, in Group B, only dihydrotestosterone was reduced in serum and penis. Histopathological examination revealed that Group C showed statistically significant histological changes, such as an increase in fibrotic tissue in the bladder, prostate, and penis. Similarly, Group B showed fibrotic changes in the prostate and penis compared with the Group A. Immunofluorescent staining revealed that the androgen receptor was more strongly expressed than the estrogen receptor beta in Group A. On the other hand, in Group C, weak expression of the androgen receptor and strong expression of the estrogen receptor beta was noted. In Group B, these changes were noted in the prostate and penis. These findings suggest that dutasteride cause morphological changes not only in prostate but also in penis. These changes are associated with altered expression patterns of androgen receptor and estrogen receptor.

Prostatorectal fistula following intravesical bacillus Calmette–Guérin immunotherapy for carcinoma in situ of the urinary bladder

Intravesical bacillus Calmette–Guérin (BCG) immunotherapy for non-muscle-invasive bladder cancer is generally regarded as a safe and effective therapy, but as BCG is a live attenuated vaccine, infectious complications occasionally occur. We report a case of prostatorectal fistula following intravesical BCG immunotherapy. A 69-year-old man with prickly sensation in the right groin presented at our hospital. Cystoscopy revealed a 2-cm papillary tumor on the trigone. Transurethral resection of the bladder tumor (TUR-BT) and bladder biopsy were performed. The histopathological finding was urothelial carcinoma, grade 2, pTa with carcinoma in situ (CIS). Then weekly intravesical BCG instillations (Connaught strain, 81 mg/50 mL) were performed. After the fifth instillation, the patient complained of continuous irritative bladder symptoms. Urine analysis showed marked pyuria, unlike the previous urine analysis. Although the BCG instillations were interrupted, the symptoms did not improve. Two weeks after the last BCG instillation, the patient suffered low-grade fever (<38°C). Ciprofloxacin was administered empirically, but after 1 week, the patient demonstrated high-grade fever (>40°C). Blood analysis showed white blood cell 24 600/mL and C-reactive protein 6.59 mg/dL. On digital rectal examination, a flat and smooth mass was palpated in the left side of the prostate without tenderness, and a CT scan confirmed swelling and the inhomogeneous enhancement of the left lobe. The physical and radiographic findings were consistent with a prostatic abscess. We discussed the case with an infectious disease consultant, and administration of trimethoprim-sulfamethoxazole was selected. Antimycobacterium antibiotics were not selected because infection with Mycobacterium bovis is very rare in the normal immune state, and the patient had been already treated with ciprofloxacin, which has an antimycobacterium effect. On its administration, the fever reduced and the irritable bladder symptoms disappeared. However, after 1 month, the abscess again increased in size and was about to rupture the rectal wall (Fig. 1a). For drainage, transurethral resection of the prostate was performed. Subsequent histology showed granulomatous prostatitis with multinucleated giant cells and caseous necrosis, and polymerase chain reaction of the specimen was positive for acid-fast bacteria. After the urethral catheter was removed, a little urine was noted as leaking from the rectum, and fistula was confirmed by cystourethrography (Fig. 1c). We discussed the need for a colostomy with surgical consultants, but chose the nonoperative course, because the fistula was small without fecaluria. The patient was given daily rifampicin, isoniazid , pyrazinamide and ethambutol. After completion of a 9-month regimen of antimycobacterial therapy, the abscess disappeared (Fig. 1b), however the patient needed another TUR-BT for the recurrent tumor. He is now in good health and voids with a good urinary stream. The prostate abscess as a complication of intravesical BCG immunotherapy is a rare condition. According to our research, only two cases of prostate abscesses have been