Hiroki Kubota

In vivo Gene Transfer into Testis and Sperm: Developments and Future Application

Despite significant advances in the treatment of infertility via assisted reproductive technology (ART), the underlying causes of idiopathic male infertility still remain unclear. Accumulating evidence suggests that disorders associated with testicular gene expression may play an important role in male infertility. To be able to fully study the molecular mechanisms underlying spermatogenesis and fertilization, it is necessary to manipulate gene expression in male germ cells. Since there is still no reliable method of recapitulating spermatogenesis culture, the development of alternative transgenic approaches is paramount in the study of gene function in testis and sperm. Established methods of creating transgenic animals rely heavily upon injection of DNA into the pronucleus or the injection of transfected embryonic stem cells into blastocysts to form chimeras. Despite the success of these two approaches for making transgenic and knockout animals, concerns remain over costs and the efficiency of transgene integration. Consequently, efforts are in hand to evaluate alternative methodologies. At present, there is much interest in developing approaches that utilize spermatozoa as vectors for gene transfer. These approaches, including testis mediated gene transfer (TMGT) and sperm mediated gene transfer (SMGT), have great potential as tools for infertility research and in the creation of transgenic animals. The aim of this short review is to briefly describe developments in this field and discuss how these gene transfer methods might be used effectively in future research and clinical arenas.

Simple Method of Preventing Postoperative Inguinal Hernia After Radical Retropubic Prostatectomy

OBJECTIVES To establish a novel and simple method of preventing post-retropubic prostatectomy (RRP) inguinal hernia. Inguinal hernias occur in 8%-22% of men within 1-2 years of RRP. Although manipulation during RRP might weaken the normal fascia structure at the internal inguinal ring with the vas deferens, the exact mechanism of post-RRP inguinal hernia remains unknown. METHODS Several surgeons performed RRP at our hospital on 271 patients between April 2004 and September 2009. Among these patients, post-RRP measures to prevent inguinal hernia were applied to 101 patients (group A) and not applied to 170 patients (group B). We released the bilateral spermatic cord from the peritoneum before suturing the wound, which should prevent the intestinal tract coated with the peritoneum from pushing through the internal inguinal tract. We compared the incidence of postoperative inguinal hernia between the 2 groups. RESULTS The patients were followed up for an average of 11.6 (range: 2-22 months) and 23.9 (range: 23-24 months) months in groups A and B, respectively. Inguinal hernia developed in no patients in group A and in 20 (11.8%) in group B. The hernia-free rate was significantly lower in group B than group A. All postoperative inguinal hernias were indirect. The median interval between surgery and hernia diagnosis was 10.6 months (range, 2-24), and 16 patients (80%) were diagnosed within 12 months. CONCLUSIONS We developed a simple method of preventing inguinal hernia after RRP. Our technique is simple enough to complete within a few minutes, and the outcome is excellent.

In Vivo Gene Transfer by Electroporation Allows Expression of a Fluorescent Transgene in Hamster Testis and Epididymal Sperm and Has No Adverse Effects upon Testicular Integrity or Sperm Quality

Abstract The study of gene function in testis and sperm has been greatly assisted by transgenic mouse models. Recently, an alternative way of expressing transgenes in mouse testis has been developed that uses electroporation to introduce transgenes into the male germ cells. This approach has been successfully used to transiently express reporter genes driven by constitutive and testis-specific promoters. It has been proposed as an alternative method for studying gene function in testis and sperm, and as a novel way to create transgenic animals. However, the low levels and transient nature of transgene expression that can be achieved using this technique have raised concerns about its practical usefulness. It has also not been demonstrated in mammals other than mice. In this study, we show for the first time that in vivo gene transfer using electroporation can be used to express a fluorescent transgene in the testis of a mammal other than mice, the Syrian golden hamster. Significantly, for the first time we demonstrate expression of a transgene in epididymal sperm using this approach. We show that expression of the transgene can be detected in sperm for as long as 60 days following gene transfer. Finally, we provide the first systematic demonstration that this technique does not lead to any significant long-term adverse effects on testicular integrity and sperm quality. This technique therefore offers a novel way to study gene function during fertilization in hamsters and may also have potential as a way of creating transgenic versions of this important model species.

Cyclooxygenase-2 protects germ cells against spermatogenesis disturbance in experimental cryptorchidism model mice.

The role of cyclooxygenases (COX) in the male reproductive organ remains unclear. However, there are some reports suggesting that COX-2 might have an effect on spermatogenesis or steroidogenesis. In this study, we examined whether COX-2 was induced in impaired testes, and we also investigated the possible role of COX in the testes using experimental cryptorchidism model mice. Five-week-old male mice underwent an operation to induce unilateral cryptorchidism via an abdominal incision and suturing of the left testes to the lateral abdominal wall, and they were then divided into 3 groups: 1) experimental cryptorchidism plus SC560 (selective COX-1 inhibitor) administration; 2) experimental cryptorchidism plus NS398 (selective COX-2 inhibitor) administration; 3) and experimental cryptorchidism alone. The expression levels of COX-1 and COX-2 were determined by immunohistologic staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR). The influence of COX inhibitors on the testes was assessed by measuring the concentration of serum testosterone and evaluating the seminiferous tubules according to the Johnsen score. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining was also performed to detect apoptosis in the testes. Immunohistologic staining and RT-PCR revealed that the expression of COX-2 was increased in the experimental cryptorchid testes (groups 1-3). The concentration of serum testosterone was significantly lower in group 2 at 5 weeks after surgery than in the other groups. The Johnsen score of the cryptorchid testes in group 2 was significantly lower than those in other groups at 5 weeks after surgery. TUNEL staining revealed that the number of apoptotic cells was significantly increased in group 2 compared with the other groups. However, the COX-1 inhibitor did not appear to affect spermatogenesis in the experimental cryptorchid testes. These results suggest that the COX-2 inhibitor provoked testicular damage in experimental cryptorchidism by inducing germ cell apoptosis. The expression of COX-2 might be induced to protect germ cells from heat stress caused by experimental cryptorchidism.

Involvement of calpain for apoptosis in dysfunction of the unaffected testis in rats with experimental testicular torsion

PROBLEM: The dynamics of calpain and involvement of apoptosis in sperm formation disorder of the unaffected testis in rats with experimental testicular torsion were investigated.
 METHODS OF STUDY: Using 6‐week‐old Wistar rats, an experimental unilateral testicular torsion model was prepared. The bilateral testes were excised 1, 3, 5, 7, 14, 35, and 70 days after the left testis was twisted, and the unaffected testes were subjected to immunohistological staining, sodium dodecyl sulfate polyacrilamide gel electrophoresis and Western blotting using anti‐calpain antibodies. Apoptosis was detected by the TdT‐mediated dUTP–biotin nick end‐labeling (TUNEL) method.
 RESULTS: By immunohistological staining, positive immunostaining by anti‐pro μ‐calpain antibody was observed in the spermatocyte nucleus, but not with anti‐pro m‐calpain antibody. The staining was increased until 7 days after testicular torsion, then decreased with progression of sperm formation disorder. By Western blotting, the intensity of staining with anti‐pro μ‐calpain antibody was increased until 7 days after torsion of the testis. Apoptosis expression in the unaffected testis was significantly inhibited by addition of a calpain inhibitor.
 CONCLUSIONS: It was suggested that μ‐calpain may be involved in apoptosis expression in sperm formation disorder of the unaffected testis in unilateral testicular torsion.

Clinical Impact of Palliative Treatment Using Octreotide for Inoperable Malignant Bowel Obstruction Caused by Advanced Urological Cancer

Malignant bowel obstruction (MBO), an occasional complication in patients with advanced urological cancer, causes gastrointestinal symptoms such as nausea and vomiting leading to suffering which severely impairs quality of life (QOL). Drug therapy, especially octreotide, a synthetic analog of somatostatin, is reportedly effective in controlling the symptoms of MBO. In the present study, we administered octreotide to urological cancer patients with MBO and evaluated the improvement of subjective symptoms, oral intake, and nasogastric intubation. Fourteen terminally ill urological cancer patients suffering with MBO were included (age range 55-92, 10 male, 4 female). Octreotide was administered at 300μg/day to those patients subcutaneously as a continuous injection. Significant improvements in subjective symptoms were observed in thirteen patients (92.8%), and ten patients (71.4%) were able to resume oral intake. Four patients required nasogastric drainage before the administration of octreotide, but nasogastric intubation was discontinued in all these cases after the use of octreotide. Early initiation of octreotide resulted in better improvement of MBO symptoms, and no adverse event was observed in any of the patients. These results revealed that 300μg/day dose of octreotide is safe and effective for managing gastrointestinal symptoms of terminally ill urological cancer patients with MBO. We also recommend starting the treatment with ocreotide as soon as MBO is diagnosed.

Spontaneous disappearance of a renal arteriovenous malformation.

Abstract We describe herein a case of complete spontaneous disappearance of a congenital arteriovenous malformation (AVM). A 28‐year‐old male was hospitalized for right flank pain and gross hematuria, followed by bladder tamponade. To improve the patients symptoms, bladder irrigation was performed. Cystoscopy demonstrated bloody urine from the right ureteral orifice. Right selective renal arteriography demonstrated tortuous, coiled vascular channels with early filling of the renal vein. Thus, right renal AVM was diagnosed. However, the patient refused further treatment and was discharged. One year later, massive hematuria recurred with bladder tamponade and the patient was rehospitalized. Renal arteriography did not show any evidence of AVM and there has been no hematuria since.

Chromosomal anomalies and sperm retrieval outcomes of patients with non‐obstructive azoospermia: a case series

Some preoperative factors affecting the outcome of microdissection testicular sperm extraction (micro‐TESE) have been previously evaluated. However, other than Klinefelter syndrome (KS), no other chromosomal anomalies have been discussed in the context of sperm retrieval outcomes. The objective of this study was to describe chromosomal anomalies and their relationship with sperm retrieval outcomes in patients with non‐obstructive azoospermia (NOA). Of the 197 NOA patients whose clinical records were retrospectively reviewed, 144 (73.1%) had normal 46,XY karyotype, 40 (20.3%) had KS (47,XXY), and 13 (6.6%) had other chromosomal anomalies (autosomal in seven cases and sex‐chromosomal anomalies in six). Of the seven patients with autosomal anomalies, two had the reportedly normal variant 46,XY,inv(9)(p12;q13). Testicular volume and serum hormone levels (luteinizing hormone, follicle‐stimulating hormone, and total testosterone) of the patients with chromosomal anomalies other than KS were comparable to those of the patients with normal karyotype. The sperm retrieval rate of the patients with 46,XY karyotype, KS, or other chromosomal anomalies were 27.1%, 22.5%, and 15.4%, respectively, with no statistically significant difference. However, among the samples collected from the 13 patients with chromosomal anomalies other than KS, only those from the two patients with the normal variant 46,XY,inv(9)(p12;q13) contained spermatozoa. Among our series of NOA patients, the incidence of autosomal anomalies was higher than that generally noted among neonates, which suggests that not only sex‐chromosomal anomalies but also autosomal anomalies may affect the development of NOA. Furthermore, our findings suggest that sperm retrieval outcome is more unfavorable in NOA patients with chromosomal anomalies than in NOA patients with 46,XY karyotype or KS, despite the use of micro‐TESE.

Outcomes of Robot-Assisted Laparoscopic Prostatectomy with a Posterior Approach to the Seminal Vesicle in 300 Patients

Background. The goal of this study was to analyze the perioperative outcomes of robot-assisted laparoscopic radical prostatectomies (RALPs) performed at our center. Methodology. We retrospectively reviewed 300 consecutive patients with clinically localized prostate cancer who underwent RALP with a posterior dissection approach to the seminal vesicle between May 2011 and November 2013. The mean patient age was 67.2 ± 5.5 years (range: 41–78 years), and the mean prostate-specific antigen (PSA) concentration, at diagnosis of prostate cancer, was 9.16 ± 6.50 ng/mL (range: 2.20–55.31 ng/mL). Results. The median duration of robotic surgery was 160 min (mean: 165 ± 40 min; range: 75–345 min). Median estimated blood loss, including that in urine, was 200 mL (mean: 277 ± 324 mL; range: 4–3250 mL). Intraoperative and immediate postoperative complications occurred in 3.0% of patients; 4 patients required allogeneic blood transfusion. As a measure of patient continence, 82.4% did not use more than 1 absorbent pad in 24 h, at 6 months postoperatively. Conclusion. RALP with an initial posterior dissection to the seminal vesicle was a safe and efficient method for controlling prostate cancer, even in these initial cases.

Human sperm motility in a microgravity environment

Background and AimsWe carried out clinostat and parabolic flight experiments to examine the effects of a microgravity (,uG) environment on human sperm motility.MethodsSemen samples were obtained manually from 18 healthy men (aged 27.4 ± 5.4 years) who had given their informed consent. In dinostat experiments, samples that were left stationary were used as a stationary control. Samples rotated vertically and horizontally were used as a rotation control and a dinostat rotation, respectively. In parabolic flight experiments using a jet plane, sperm motility was compared for each parameter at μG, 1G and 2G. The state of 1G during the flight was used as a control. Sperm motility was determined using an automatic motility analyzer HT-M2030 in a microgravity environment.ResultsAll parameters of sperm motility tended to be lower in dinostat rotation compared with rotation control at both low-speed and high-speed, but the differences were not statistically significant. In parabolic flight, sperm motility and parameters of linear movement were decreased (P< 0.05). There was no significant difference between μG and 2G, but sperm motility was significantly decreased at μG than at 1G.ConclusionsOur findings suggest that sperm motility is reduced under μG.