[Show abstract][Hide abstract] ABSTRACT: Cancer treatments, either chemo- or radiotherapy, may cause severe damage to gonads which could lead to the infertility of patients. In post-pubertal male patients, semen cryopreservation is recommended to preserve the potential to have their own biological children in the future; however, it is not applicable to prepubertals. The preservation of testis tissue which contains spermatogonial stem cells (SSCs) but not sperm would be an alternative measure. The tissues or SSCs have to be transplanted back into patients to obtain sperm; however, this procedure remains experimental, invasive, and is accompanied with the potential risk of re-implantation of cancer cells. Recently, we developed an organ culture system which supports the spermatogenesis of mice up to sperm formation from SSCs. It was also shown that the tissues could be frozen for later sperm production, which resulted in the generation of offspring. Thus, it could be useful as a clinical application for preserving the reproductive potential of male pediatric cancer patients. The establishment of an optimized cryopreservation method and the development of a culture system for human testis tissue are expected in the future.
Preview · Article · Aug 2015 · Reproductive Medicine and Biology
[Show abstract][Hide abstract] ABSTRACT: Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.
[Show abstract][Hide abstract] ABSTRACT: Twenty years ago, the transplantation of spermatogonial stem cells (SSCs) from a mouse to other recipient mice was shown to be feasible, which clearly demonstrated the functional identity of SSCs. Since then, several important new findings and other technical developments have followed, which included a new hypothesis on their cell kinetics and spermatogonial hierarchy in the testis, a culture method allowing their self-renewal and proliferation, a testis tissue organ culture method, which induced their complete differentiation up to sperm, and the in vitro induction of germ cells from embryonic stem cells and induced pluripotent stem cells. These advancements reinforced or advanced our understanding of this unique cell. Nonetheless, there are many unresolved questions in the study of spermatogonial stem cells and a long road remains until these cells can be used clinically in reproductive medicine.
Preview · Article · May 2015 · Asian Journal of Andrology
[Show abstract][Hide abstract] ABSTRACT: With the increasing cure rate of paediatric cancers, infertility, as one of the adverse effects of treatments, has become an important concern for patients and their families. Since semen cryopreservation is applicable only for post-pubertal patients, alternative pre-pubertal measures are necessary. Here we demonstrate that testis tissue cryopreservation is a realistic measure for preserving the fertility of an individual. Testis tissues of neonatal mice were cryopreserved either by slow freezing or by vitrification. After thawing, they were cultured on agarose gel and showed spermatogenesis up to sperm formation. Microinsemination was performed with round spermatids and sperm, leading to eight offspring in total. They grew healthily and produced progeny upon natural mating between them. This strategy, the cryopreservation of testis tissues followed by in vitro spermatogenesis, is promising to preserve the fertility of male paediatric cancer patients in the future.
[Show abstract][Hide abstract] ABSTRACT: The in vitro propagation of mouse spermatogonial stem cells (SSCs) became possible in 2003; these cultured SSCs were named germ-line stem (GS) cells. To date, however, it has not been possible to induce spermatogenesis from GS cells in vitro. Recently, we succeeded in producing functional sperm from primitive spermatogonia in explanted neonatal mouse testis tissues. Here we describe a protocol that can support spermatogenesis from GS cells up to sperm formation in vitro using an organ culture method. GS cells transplanted in the extracted testis form colonies in the tissue fragments and differentiate into sperm under the described in vitro organ culture conditions. It takes about 6 weeks to obtain sperm from GS cells. The sperm are viable, resulting in healthy offspring through micro-insemination. Thus, this protocol should be a valuable tool for the study of mammalian spermatogenesis.
[Show abstract][Hide abstract] ABSTRACT: It is a challenge to induce spermatogenesis in vitro, because it involves a complicated process of sequential cell proliferation and differentiation, from spermatogonial stem cells to sperm formation. Recently, we have succeeded, using a classical organ culture method, to produce sperm from spermatogonial stem cells, using several modifications. The produced spermatids and sperm were fertile, giving rise to healthy progeny. This new in vitro system for spermatogenesis could be useful for addressing the issue of sperm quality that is becoming more important in this ICSI era. We also hope that an in vitro system of human spermatogenesis will be developed in the near future.
No preview · Article · Oct 2013 · Journal of Mammalian Ova Research
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Azoospermia is a common side effect of chemotherapy. Although most patients restore spermatogenesis over time, the exact time course has not been well described. We analyzed the recovery of spermatogenesis in testicular cancer patients following chemotherapy.
Patients and methods:
49 patients, consisting of 45 treated with a bleomycin, etoposide and cisplatin (BEP) regimen and 4 with high-dose chemotherapy, were followed up with occasional semen analyses. The primary endpoint of this study was the confirmation of motile spermatozoa in the patients' semen.
Among 45 patients treated with BEP, 44 recovered spermatogenesis. The recovery of spermatogenesis was delayed depending on the increase in BEP cycles. In groups of patients who received 1-2, 3 and 4 cycles, the recovery rates of spermatogenesis within 2 year were 83.3, 80.0 and 66.7%, respectively. In the group with 5-6 cycles of BEP, re-spermatogenesis was significantly delayed and no patients re-established spermatogenesis within 2 years. The patients' age and semen parameters before chemotherapy were not useful as predictive factors for the recovery of spermatogenesis.
The recovery of spermatogenesis was rather fast and was often observed as early as several months after BEP treatment when the number of cycles was <4.
No preview · Article · Sep 2013 · Urologia Internationalis
[Show abstract][Hide abstract] ABSTRACT: The trans-Golgi-network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 was detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting revealed that SMAP2-deficient male mice were healthy and survived to adulthood, but were infertile and exhibited globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increased, TGN structures were distorted, acrosome formation was severely impaired, and reorganization of the nucleus did not proceed properly. CALM functions to regulate vesicle sizes, and this study showed that CALM was not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the SNARE complex, was not properly concentrated at the site of acrosome formation. Thus, the present study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.
Full-text · Article · Jul 2013 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: It is known that cells of testis tissues in fetal or neonatal periods have the ability to reconstruct the testicular architecture even after dissociation into single cells. This ability, however, has not been demonstrated effectively in vitro. In our present study, we succeeded in reconstructing seminiferous tubules in vitro which supported spermatogenesis to meiotic phase. Testis cells of neonatal mice were dissociated enzymatically into single cells. The cells formed aggregates in suspension culture and were transferred to the surface of agarose gel to continue the culture with a gas-liquid interphase method, where a tubular architecture gradually developed during the following 2 weeks. Immunohistological examination confirmed Sertoli cells forming tubules and germ cells inside. With testis tissues of Acr-GFP transgenic mice, whose germ cells express GFP during meiosis, cell aggregates formed a tubular structure and showed GFP expressions in their reconstructed tissues. Meiotic figures were also confirmed by regular histology and immunohistochemistry. In addition, we mixed cell lines of spermagonial stem cells (GS cells) into the testis cell suspension, and found the incorporation of GS cells in the tubules in reconstructed tissues. When GS cells derived from Acr-GFP transgenic mice were used, GFP expression was observed, indicating that the spermatogenesis of GS cells was proceeding up to the meiotic phase. This in vitro reconstruction technique will be a useful method for the study of testis organogenesis and spermatogenesis.
Preview · Article · Jun 2013 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: To retrospectively assess the clinical utility in ureteroscopy (URS) planning of cumulative stone diameter (CSD), which does not account for stone width or depth, as a predictor of URS outcome and compare it with stone volume.
Patients with renal stones treated at a single institute by flexible URS were retrospectively evaluated. To assess the clinical utility of CSD, relationships between stone-free (SF) status and stone burden (CSD and volume) were analyzed using the area under the receiver operating characteristics (AUROC) curve. To identify stone number impact on CSD, the AUROC of CSD divided by stone number was evaluated. Correlation coefficients of CSD and stone volume were also calculated for groups by stone number.
In cases with CSD <20.0 mm, CSD and stone volume revealed equal ability to predict SF status. In cases with CSD ≥20.0 mm, stone volume showed higher predictive ability. The ROC curves for cases with ≥4 stones showed that CSD was less predictive of SF status than stone volume. The correlation coefficients of CSD and stone volume by stone number were 0.922 for 1 stone, 0.900 for 2-3 stones, and 0.661 for ≥4 stones.
In cases with CSD ≥20.0 mm or ≥4 stones, we should evaluate stone volume for a more predictive stone burden, and pretreatment non-contrast CT seems sufficient. In cases with CSD <20.0 mm or 1-3 stones, CSD was as valid a predictor of preoperative stone burden as stone volume, so preoperative kidney-ureter-bladder (KUB) films may be sufficient.
[Show abstract][Hide abstract] ABSTRACT: We investigated the Core Lower Urinary Tract Symptom Score as an outcome assessment tool for the treatment of lower urinary tract symptoms using silodosin. In addition, the ability of the Core Lower Urinary Tract Symptom Score to detect overactive bladder in male patients with lower urinary tract symptoms was examined. The present study included 241 males with benign prostatic hyperplasia treated at 31 medical facilities between June 2009 and December 2010. All patients were given silodosin, and the effects of silodosin intake were measured using four questionnaires: the Core Lower Urinary Tract Symptom Score, International Prostate Symptom Score, Overactive Bladder Symptom Score and Quality-of-Life index. The efficacy of silodosin for treating lower urinary tract symptoms was validated according to the total scores of all four questionnaires weighted equally (P < 0.05). Spearman's ρ among the Core Lower Urinary Tract Symptom Score, International Prostate Symptom Score and Overactive Bladder Symptom Score showed a mild-high correlation. However, the correlation between the baseline values of the Core Lower Urinary Tract Symptom Score and Quality-of-Life index was low in the groups with benign prostatic hyperplasia (ρ = 0.314) and benign prostatic hyperplasia/overactive bladder (ρ = 0.244). Our findings showed the Core Lower Urinary Tract Symptom Score, both its total score and each subscore, is able to show the efficacy of silodosin, similar to other questionnaires. The Core Lower Urinary Tract Symptom Score is also useful for identifying overactive bladder symptoms in patients with benign prostatic hyperplasia. As the Core Lower Urinary Tract Symptom Score does not correlate well with the Quality-of-Life index, these two questionnaires might be better used in combination to assess treatment outcomes.
Full-text · Article · May 2013 · International Journal of Urology
[Show abstract][Hide abstract] ABSTRACT: This study investigated the correlation between the operation time using two different power settings of a Ho: YAG laser.
A total of 68 patients underwent cystolithotripsy from April 2010 to October 2011 In Fifty-six of these patients underwent cystolithotripsy by one surgeon using a Ho: YAG laser for bladder calculi. This study assessed these patients in two groups; the 30 W laser generator group with the settings of 2.5 J x 5 Hz (30 W group) and the 100 W laser generator group as the settings of 3.5 J x 5 Hz (100 W group). The operation time in these two groups were assessed.A total of 56 patients including 45 male and 11 female patients that underwent cystolithotripsy using a Ho: YAG laser for bladder calculi by one surgeon were enrolled in this study. The patients' characteristics including age (mean; 68.8 vs 68.4 yr), gender (male; 74.2 vs 88.0%), stone burden (mean; 34.9 vs 41.3 mm), number of stones (mean; 3.2 vs 2.0) and stone's CT density (mean; 981.5 vs 902.0 HU) showed no significant differences. All patients were stone free following treatment. The median total length of the operation was 19 minutes (mean: 34.6 ± 36.1) in the 30 W group and 29 minutes (mean: 44.4 ± 38.8) in the 100 W group, which was not significantly different.
The results showed that the power settings of Ho: YAG laser show no differences in the operation time for bladder calculi lithotripsy.
Full-text · Article · Mar 2013 · BMC Research Notes
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To investigate the utility and limitations of stone surface area (SA) as a predictor of stone-free (SF) status after a single semirigid ureteroscopy (URS), with or without a flexible component, for the treatment of patients with urinary stones.
Patients and methods:
Cases of patients with urinary stones treated by combined URS with holmium laser lithotripsy at a single institute were retrospectively evaluated. Correlations of possible predictors with SF status were analyzed using a logistic regression model. Two types of SA were measured: "Traced stone surface area" (tSA) and "calculated stone surface area" (cSA).
According to the univariate analysis, the following variables were significantly associated with non-SF status: Stone number (P<0.001), ureteral stone location (P=0.045), presence of renal stones (P<0.001), tSA (P<0.001), cSA (P<0.001), stone volume (P<0.001), and operator experience (P=0.02). According to multivariate analysis, stone volume (P=0.016) was an independent predictor of SF status. The scatter diagrams for tSA and cSA showed strong correlations between these parameters, and Spearman ρ was 0.975.
Stone volume and SA were highly indicative of stone status after single semirigid URS, with or without a flexible component. The formula for cSA, maximum diameter×width×π×1/4, was demonstrated to accurately represent SA in this study. SA, however, indicated a lower clinical priority and utility as a predictor of stone status than stone volume. The combination of semirigid and flexible URS could access any ureteral stones, including those that semirigid URS alone could not treat. The cutoff points for these predictors of outcome were 110.0 mm(2) for cSA, 125.0 mm(2) for tSA, and 840.0 mm(3) for stone volume.
Full-text · Article · Feb 2013 · Journal of endourology / Endourological Society
[Show abstract][Hide abstract] ABSTRACT: Research on in vitro spermatogenesis has a long history and remained to be an unaccomplished task until very recently. In 2010, we succeeded in producing murine sperm from primitive spermatogonia using an organ culture method. The fertility of the sperm or haploid spermatids was demonstrated by microinsemination. This organ culture technique uses the classical air-liquid interphase method and is based on conditions extensively examined by Steinbergers in 1960s. Among adaptations in the new culture system, application of serum-free media was the most important. The system is very simple and easy to follow.
Full-text · Article · Jan 2013 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Objective:
To evaluate the effectiveness of cystoscopic lithotripsy, we performed Amplatz sheath technique using Ho: YAG laser. Maheshwari first reported the use of an Amplatz sheath in the female urethra in 1998, and Okeke et al reported the use of an Amplatz sheath for male patients during cystolithotripsy in 2004. The usefulness of the holmium (Ho): yttrium aluminum garnet (YAG) laser lithotripsy is widely accepted, even for large bladder calculi. Since then, there have been no more reports of using the sheath with an Ho: YAG laser.
We inserted the Amplatz sheath conversely. Because of the clear visualization, we used higher laser settings with 2.5 J × 15 to 20 Hz.
We experienced 3 female patients that were successfully treated with the Amplatz sheath technique using Ho: YAG laser lithotripsy. In these 3 patients, whose stone burdens were 4.5, 3.8, and 4.3 cm, they were able to successfully become stone-free with surgeries of 74 minutes, 67 minutes, and 58 minutes, respectively, with no complications.
We experienced 3 female patients that were successfully treated with the Amplatz sheath technique using Ho: YAG laser lithotripsy.
[Show abstract][Hide abstract] ABSTRACT: Male infertility is most commonly caused by spermatogenic defects or insufficiencies, the majority of which are as yet cureless. Recently, we succeeded in cultivating mouse testicular tissues for producing fertile sperm from spermatogonial stem cells. Here, we show that one of the most severe types of spermatogenic defect mutant can be treated by the culture method without any genetic manipulations. The Sl/Sl(d) mouse is used as a model of such male infertility. The testis of the Sl/Sl(d) mouse has only primitive spermatogonia as germ cells, lacking any sign of spermatogenesis owing to mutations of the c-kit ligand (KITL) gene that cause the loss of membrane-bound-type KITL from the surface of Sertoli cells. To compensate for the deficit, we cultured testis tissues of Sl/Sl(d) mice with a medium containing recombinant KITL and found that it induced the differentiation of spermatogonia up to the end of meiosis. We further discovered that colony stimulating factor-1 (CSF-1) enhances the effect of KITL and promotes spermatogenesis up to the production of sperm. Microinsemination of haploid cells resulted in delivery of healthy offspring. This study demonstrated that spermatogenic impairments can be treated in vitro with the supplementation of certain factors or substances that are insufficient in the original testes.
Full-text · Article · Sep 2012 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Complete staghorn calculi are typically managed with percutaneous nephrolithotomy (PCNL). However, dilating nephrostomy and inserting a nephro access sheath can be difficult to perform without hydronephrosis. We reported the procedure of ureteroscopy-assisted retrograde nephrostomy (UARN) during PCNL. UARN is effective without dilating the renal collecting system in cases of complete staghorn calculi. A 63-year old female with a left complete staghorn renal calculus was referred to our hospital. Under general and epidural anesthesia, the patient was placed in a modified-Valdivia position. A flexible ureteroscope was inserted and a Lawson retrograde nephrostomy puncture wire was advanced into the flexible ureteroscope. The puncture wire was forwarded along the route from the renal pelvis to the exit skin. Calculus fragmentation was done using a pneumatic lithotripter and the Ho: YAG laser. UARN during PCNL was effective for the treatment of a complete staghorn calculus.