[Reconstruction of spermatogonial niche for male fertility preservation].

Infertility is one of the late side effects of cancer treatment. Expansion of anti-cancer treatment allow patients to have more life time, however infertility is becoming a matter damaging QOL during the young cancer survivors. The passive strategy such as avoiding the gonad-toxic drug or decreasing the total volume of them and shielding the gonads against cancer therapy has been conducted. To preserve the fertility of young female, ovary tissue cryopreservation is becoming a standard over the world after the success of offspring from cryopreserved ovary tissue autograft was reported. Sperm preservation method is established for the male fertility preservation method, however this is only applicable for sexually matured male patients. For the sake of preserving fertility of sexually immature male patients, many trials using cryopreserved testis tissues or testicular cells have been undergone. Recently, in vitro gametogenesis from stem cell of the human and the mouse to primordial germ cell like cell has been achieved. Here the previous challenges and the latest reports for obtaining functional sperm from immature testis and the reconstruction of spermatogonial niche as a potential approach for preserving fertility procedure are described.

Differentiation of fetal sertoli cells in the adult testis.

Sertoli cells proliferate and construct seminiferous tubules during fetal life, then undergo differentiation and maturation in the prepubertal testes. In the adult testes, mature Sertoli cells maintain spermatogonia and support spermatogenesis during the entire lifetime. Although Sertoli-like cells have been derived from iPS cells, they tend to remain immature. To investigate whether Sertoli cells can spontaneously acquire the ability to support spermatogenesis when transferred into the adult testis, we transplanted mouse fetal testicular cells into a Sertoli-depleted adult testis. We found that donor E12.5, E14.5 and E16.5 Sertoli cells colonized adult seminiferous tubules and supported host spermatogenesis 2 months after transplantation, demonstrating that immature fetal Sertoli cells can undergo sufficient maturation in the adult testis to become functional. This technique will be useful to analyze the developmental process of Sertoli cell maturation and to investigate the potential of iPS-derived Sertoli cells to colonize, undergo maturation, and support spermatogenesis within the testis environment.

A transgenic DND1GFP fusion allele reports in vivo expression and RNA-binding targets in undifferentiated mouse germ cells†.

In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

Sertoli cell ablation and replacement of the spermatogonial niche in mouse.

Spermatogonia, which produce sperm throughout the male lifetime, are regulated inside a niche composed of Sertoli cells, and other testis cell types. Defects in Sertoli cells often lead to infertility, but replacement of defective cells has been limited by the inability to deplete the existing population. Here, we use an FDA-approved non-toxic drug, benzalkonium chloride (BC), to deplete testis cell types in vivo. Four days after BC administration, Sertoli cells are preferentially depleted, and can be replaced to promote spermatogenesis from surviving (host) spermatogonia. Seven days after BC treatment, multiple cell types can be engrafted from fresh or cryopreserved testicular cells, leading to complete spermatogenesis from donor cells. These methods will be valuable for investigation of niche-supporting cell interactions, have the potential to lead to a therapy for idiopathic male infertility in the clinic, and could open the door to production of sperm from other species in the mouse.

Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device.

In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole.

Cryopreservation of testis tissues and in vitro spermatogenesis.

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.

Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9.

Mouse spermatogonial stem cells (SSCs) can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS) cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure.

Offspring production with sperm grown in vitro from cryopreserved testis tissues.

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.

In Vitro Reconstruction of Mouse Seminiferous Tubules Supporting Germ Cell Differentiation.

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.

In vitro spermatogenesis using an organ culture technique.

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.

Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice.

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.

[Three dysuria adults without continuous medical treatment after surgery for spina bifida in infancy].

We encountered three patients with dysuria who had undergone spinal surgery for spina bifida during infancy. The patients presented with mental disability and dysbasia. Difficulty in urination, urinary incontinence, and a residual sensation of urine were resolved through intermittent self-catheterization in all patients. It was speculated that treatment for dysuria in these patients was delayed because they were not aware of its association with their condition during infancy, dysuria was relatively mild, and they had no history of febrile urinary tract infection. It is important for attending physicians to explain to parents of such infants the possible association of spina bifida with the future risk of dysuria, and to consider long-term follow-up to monitor their outcome.

In vitro production of fertile sperm from murine spermatogonial stem cell lines.

Spermatogonial stem cells (SSCs) are the only stem cells in the body that transmit genetic information to the next generation. The long-term propagation of rodent SSCs is now possible in vitro, and their genetic modification is feasible. However, their differentiation into sperm is possible only under in vivo conditions. Here we show a new in vitro system that can induce full spermatogenesis from SSC lines or any isolated SSCs. The method depends on an organ culture system onto which SSCs are transplanted. The settled SSCs form colonies and differentiate up into sperm. The resultant haploid cells are fertile, and give rise to healthy offspring through micro-insemination. In addition, the system can induce spermatogenesis from SSCs that show spermatogenic failure due to a micro-environmental defect in their original testes. Thus, an in vitro system is established that can be used to correct or manipulate the micro-environmental conditions required for proper spermatogenesis from murine SSC lines.

[Pancreatic metastasis from renal cell carcinoma 25 years after radical nephrectomy].

We report a case of pancreatic metastasis from renal cell carcinoma detected 25 years after radical nephrectomy. A 74-year-old man, who had undergone radical nephrectomy for renal cell carcinoma at age 49, was found by computed tomography to have a strongly enhanced mass on the pancreatic head. The patient underwent pancreaticoduodenectomy and the pathological diagnosis was metastatic renal cell carcinoma. This was evidently a slow growing tumor because the metastatic pancreas tumor was well demarcated and the metastasis was found 25 years after the primary operation. Aggressive surgical treatment of isolated metastatic lesions offers a chance of long-term survival. Patients with a history of RCC should undergo a long-term follow-up to detect and evaluate metastasis to pancreas as well as other organs.

[A case of giant retroperitoneal paraganglioma].

Paraganglioma is a rare neuroendocrine tumor which arises from extra adrenal paraganglionic cells of the autonomic nervous system. We report a case of a giant retroperitoneal paraganglioma. A 43-year-old man referred to our hospital for further examination of a retroperitoneal mass. The patient had neither familial nor past medical history. The blood and urine test, laboratory examinations including cathecolamines, were unremarkable. Abdominal computed tomography showed an enhancing solid mass 13 cm in diameter on the left kidney. The invasion to the left kidney was suspected. Angiography showed the left renal, splenic, middle suprarenal and left inferior diaphragmatic artery feeding the tumor. The splenic and left inferior diaphragmatic artery were embolized before surgical treatment. The tumor, left kidney, adrenal gland and spleen were surgically resected. Histological examination revealed extra-adrenal paraganglioma, and there was no invasion of the tumor to the left kidney, adrenal gland and spleen. The patient has now survived more than 10 months following the surgery without tumor recurrence.

[ureterocele prolapse through the urethra: a case report].

We report a case of prolapse of a simple ureterocele presenting as perineural tumor. A 60-year-old woman presented with perineum pain and bleeding. A physical examination revealed a hard mass, 30 mm in diameter protruding from the external meatus. The computerized tomography, magnetic resonance imaging and cystography showed an uncharacterized tumor. Endoscopic examination was performed. However, just before resection the mass collapsed spontaneously and turned out to be a prolapse of ureterocele. No transurethral incision was performed. Eleven months postoperatively, the patient has not developed vesicoureteral reflux or urinary tract infection. Physicians should consider prolapse of a simple ureterocele in the differential diagnosis of the female meatal tumor.

[Verrucous carcinoma of penis: a case report].

We report a case of verrucous carcinoma of the penis. A 62-year-old man, who presented with penile swelling and pain, was referred to our hospital. Although, penile tumor biopsy revealed no evidence of malignancy, the patient presented with penile swelling and discharge. The penis was surgically resected and urinary diversion was performed. The pathological examination of the resected glans revealed verrucous carcinoma of penis. Furthermore, in situ hybridization revealed human papilloma virus (HPV) infection. This clearly showed that the verrucous carcinoma of the penis resulted from the HPV infection. The patient has survived for 14 months after surgery without local recurrence or metastasis.

[Low-dose docetaxel, estramustine and dexamethasone combination chemotherapy for hormone-refractory prostate cancer].

The objective of this study was to evaluate the efficacy and safety of low-dose docetaxel, estramustine and dexamethasone combination chemotherapy in patients with hormone-refractory prostate cancer (HRPC). Sixty-nine patients with HRPC were enrolled. Docetaxel was given at a dose of 25 mg/m(2) on days 1 and 8 every 3 weeks, oral estramustine 280 mg twice daily on days 1 to 3 and 8 to 10, and oral dexamethasone 1 mg daily throughout the course. Cycles were repeated every 21 days. Treatment was continued until disease progression or excessive toxicity. Patients were evaluated for response and toxicity. Patients received a median of eleven cycles (range : 1-25). Prostatic-specific antigen (PSA) was decreased greater than 50% in 53 (77%) out of 69 patients and median duration of PSA response was 10.2 months. Median time to progression and overall survival 10.2 and 24 months, respectively. Grade 1-2 fatigue was the most common toxicity observed in 10 (15%) patients. Grade 3-4 toxicities were observed in five (7%) patients (2 thrombosis, 2 bilirubin elevation, and 1 aspartate transaminase/alanine transaminase elevation). Low-dose docetaxel, estramustine and dexamethasone combination chemotherapy is an effective and well tolerated treatment for Japanese HRPC patients.

[Eosinophilic cystitis caused by Seijo-bohhu-tou: a case report].

A 19-year-old woman was admitted to our hospital with a complaint of residual feeling, frequency and pain on urination. Laboratory analysis revealed an elevated eosinophilia count in peripheral blood and white blood cell count in urine. Lymphocyte stimulation test of Chinese herb named "Seijoh-bohhuh-toh" showed a positive reaction. Bladder symptoms were improved after ceasing this Chinese herb. From these points, we considered that the Chinese herb might have caused eosinophilic cystitis. We report this rare case with a review of the literature.