Improvements in in vitro spermatogenesis: oxygen concentration, antioxidants, tissue-form design, and space control.

Incorporation of bovine serum-derived albumin formulation (AlbuMAX) into a basic culture medium, MEMα, enables the completion of in vitro spermatogenesis through testicular tissue culture in mice. However, this medium was not effective in other animals. Therefore, we sought an alternative approach for in vitro spermatogenesis using a synthetic medium without AlbuMAX and aimed to identify its essential components. In addition to factors known to be important for spermatogenesis, such as retinoic acid and reproductive hormones, we found that antioxidants (vitamin E, vitamin C, and glutathione) and lysophospholipids are vital for in vitro spermatogenesis. Moreover, based on our experience with microfluidic devices (MFD), we developed an alternative approach, the PDMS-ceiling method (PC method), which involves simply covering the tissue with a flat chip made of PDMS, a silicone resin material used in MFD. The PC method, while straightforward, integrates the advantages of MFD, enabling improved and uniform oxygen and nutrient supply via tissue flattening. Furthermore, our studies underscored the significance of lowering the oxygen concentration to 10-15%. Using an integrated cultivation method based on these findings, we successfully achieved in vitro spermatogenesis in rats, which has been a long-standing challenge. Further improvements in culture conditions would pave the way for spermatogenesis completion in diverse animal species.

A novel alternative method for long-term evaluation of male reproductive toxicity and its recovery using a pre-pubertal mouse testis organ culture system.

Successful treatment of pediatric cancers often results in long-term health complications, including potential effects on fertility. Therefore, assessing the male reproductive toxicity of anti-cancer drug treatments and the potential for recovery is of paramount importance. However, in vivo evaluations are time-intensive and require large numbers of animals. To overcome these constraints, we utilized an innovative organ culture system that supports long-term spermatogenesis by placing the testis tissue between a base agarose gel and a polydimethylsiloxane ceiling, effectively mirroring the in vivo testicular environment. The present study aimed to determine the efficacy of this organ culture system for accurately assessing testicular toxicity induced by cisplatin, using acrosin-green fluorescent protein (GFP) transgenic neonatal mouse testes. The testis fragments were treated with different concentrations of cisplatin-containing medium for 24 h and incubated in fresh medium for up to 70 days. The changes in tissue volume and GFP fluorescence over time were evaluated to monitor the progression of spermatogenesis, in addition to the corresponding histopathology. Cisplatin treatment caused tissue volume shrinkage and reduced GFP fluorescence in a concentration-dependent manner. Recovery from testicular toxicity was also dependent on the concentration of cisplatin received. The results demonstrated that this novel in vitro system can be a faithful replacement for animal experiments to assess the testicular toxicity of anti-cancer drugs and their reversibility, providing a useful method for drug development.

Sertoli cell replacement in explanted mouse testis tissue supporting host spermatogenesis†.

Spermatogenesis takes place in the seminiferous tubules, starting from the spermatogonial stem cell and maturing into sperm through multiple stages of cell differentiation. Sertoli cells, the main somatic cell constituting the seminiferous tubule, are in close contact with every germ cell and play pivotal roles in the progression of spermatogenesis. In this study, we developed an in vitro Sertoli cell replacement method by combining an organ culture technique and a toxin receptor-mediated cell knockout system. We used Amh-diphtheria toxin receptor transgenic mice, whose Sertoli cells specifically express human diphtheria toxin receptor, which renders them sensitive to diphtheria toxin. An immature Amh-diphtheria toxin receptor testis was transplanted with the donor testis cells followed by culturing in a medium containing diphtheria toxin. This procedure successfully replaced the original Sertoli cells with the transplanted Sertoli cells, and spermatogenesis originating from resident germ cells was confirmed. In addition, Sertoli cells in the mouse testis tissues were replaced by transplanted rat Sertoli cells within culture conditions without requiring immunosuppressive treatments. This method works as a functional assay system, making it possible to evaluate any cells that might function as Sertoli cells. It would also be possible to investigate interactions between Sertoli and germ cells more closely, providing a new platform for the study of spermatogenesis and its impairments.

Antioxidant vitamins and lysophospholipids are critical for inducing mouse spermatogenesis under organ culture conditions.

In vitro mouse spermatogenesis using a classical organ culture method became possible by supplementing basal culture medium with only the product of bovine serum albumin purified by chromatography (AlbuMAX), which indicated that AlbuMAX contained every chemical factor necessary for mouse spermatogenesis. However, since the identity of these factors was unclear, improvements in culture media and our understanding of the nutritional and signal substances required for spermatogenesis were hindered. In the present study, chemically defined media (CDM) without AlbuMAX was used to evaluate each supplementary factor and their combinations for the induction of spermatogenesis. Similar to in vivo conditions, retinoic acid, triiodothyronine (T3 ), and testosterone (T) were needed. Based on differences in spermatogenic competence between AlbuMAX, fetal bovine serum, and adult bovine serum, we identified α-tocopherol, which strongly promoted spermatogenesis when combined with ascorbic acid and glutathione. Differences were also observed in the abilities of lipids extracted from AlbuMAX using two different methods to induce spermatogenesis. This led to the identification of lysophospholipids, particularly lysophosphatidylcholine, lysophosphatidic acid, and lysophosphatidylserine, as important molecules for spermatogenesis. New CDM formulated based on these results induced and promoted spermatogenesis as efficiently as AlbuMAX-containing medium. In vitro spermatogenesis with CDM may provide a unique experimental system for research on spermatogenesis that cannot be performed in in vivo experiments.

Gap between UAS and ureteroscope predicts renal stone-free rate after flexible ureteroscopy with the fragmentation technique.

PURPOSE: To assess the effect of our new classification on surgical outcomes after flexible ureteroscopy (fURS) for kidney stones. METHODS: We retrospectively examined 128 patients after single renal fURS procedures performed using ureteral access sheaths (UASs) with the fragmentation technique. Based on the gap (calculated by subtracting the ureteroscope diameter from the UAS diameter), enrolled patients were divided into three groups: small (< 0.6 mm), medium (0.6 to < 1.2 mm), and large space groups (≥ 1.2 mm). Stone-free (SF) status was defined as either complete absence of stones (SF) or the presence of stones < 4 mm in diameter on non-contrast computed tomography (NCCT). RESULTS: The SF rate was significantly lower in the small space group (50% in small, 97.9% in medium, 89.2% in large; p = 0.001). Perioperative complications over Clavien-Dindo Grade I were observed in 16.7%, 4.2%, and 8.1% of patients, respectively (p = 0.452). The ratio of stone volume and operative time (efficiency of stone removal) was significantly higher in the large space group compared to the small and medium space groups (0.009 ± 0.003 ml/min, 0.013 ± 0.005 ml/min, 0.027 ± 0.012 ml/min, respectively; p < 0.001). CONCLUSION: Our findings that gaps > 0.6 mm (1.8 Fr), including the combination of a 9.5-Fr UAS and a small caliber ureteroscope, improve SF rates, and larger gaps facilitate stone removal efficiency providing the basis for future development of clinical protocols aimed at improving outcomes.

Transcriptome analysis reveals inadequate spermatogenesis and immediate radical immune reactions during organ culture in vitro spermatogenesis.

Cultivation of neonatal mouse testis tissue can induce spermatogenesis and produce fertile sperms. However, in vitro spermatogenesis mediated by the current organ culture method comes short in fully mimicking the in vivo counterpart, partly due to a lack of knowledge underlying molecular phenotypes of in vitro spermatogenesis. In this study, we investigated transcriptome of cultured testis tissues using microarray method. Principle component analysis of the transcriptome data revealed delay and/or arrest of spermatogenesis and immediate radical immune reactions in the cultured testis tissues. The delay/arrest of spermatogenesis occurred before and during early meiotic phase, resulting in inefficient progression of meiosis. The immune reaction, on the other hand, was drastic and overwhelming, in which TLR4-NF-kB signaling was speculated to be involved. Notably, treatment with TAK242, an inhibitor of TLR4-NF-kB signaling pathway, ameliorated the macrophage activation which otherwise would exacerbate the inflammation. Thus, the present study revealed for the first time at molecular level that the deficiency of germ cell differentiation and the immense immune reaction are major abnormalities in the cultured testis tissues.

Development and internal validation of a nomogram to predict perioperative complications after flexible ureteroscopy for renal stones in overnight ureteral catheterization cases.

PURPOSE: To identify risk factors by developing and internally validating a nomogram for preventing perioperative complications in overnight ureteral catheterization cases after fURS for kidney stones. METHODS: We retrospectively examined 309 patients with overnight ureteral catheterization after single fURS procedures for renal stones. fURS procedures were performed based on the fragmentation technique. The ureteral catheter was removed on postoperative day 1. Within this group, patients who experienced perioperative complications (complication group) were compared with those who did not experience complications (non-complication group). The complication group included 77 patients whose Clavien-Dindo classification score was I, II, III, or IV and/or those whose body temperature during hospitalization was over 37.5 °C. RESULTS: The overall stone volume, stone-free rate, incidence of perioperative complications, and procedure duration were 1.39 mL, 94.8%, 24.9%, and 62 min, respectively. Severe complications of a Clavien-Dindo level III or IV were observed in only four cases (1.3%). Multivariate assessment revealed five independent predictors of perioperative complications after fURS with overnight catheterization: age (p = 0.11), sex (p = 0.067), stone volume (p = 0.33), Hounsfield units (p = 0.16), and narrow ureter (p = 0.018). We developed a nomogram to predict perioperative complications after fURS using these parameters. CONCLUSIONS: We developed a predictive model for perioperative complications of patients with overnight catheterization after fURS for renal stones. This model could select patients who were at a low risk of complications.

In vitro spermatogenesis in two-dimensionally spread mouse testis tissues.

PURPOSE: Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients. METHODS: Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of Acr-Gfp, which reflects the progression of spermatogenesis. RESULTS: Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids. CONCLUSIONS: The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.

A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility.

In our previous study, we produced a microfluidic device (MFD) which successfully maintained spermatogenesis for over 6 months in mouse testis tissues loaded in the device. In the present study, we developed a new MFD, a monolayer device (ML-D) with a barrier structure consisting of pillars and slits, which is simpler in design and easier to make. This ML-D was also effective for inducing mouse spermatogenesis and maintained it for a longer period than the conventional culture method. In addition, we devised a way of introducing sample tissue into the device during its production, just before bonding the upper layer of polydimethylsiloxane (PDMS) and bottom glass slide. The tissue can obtain nutrients horizontally from the medium running beside it and oxygen vertically from above through PDMS. In addition, the glass slide set at the bottom improved the visibility of the sample tissue with an inverted microscope. When we took photos of cultured tissue of the Acr-Gfp transgenic mouse testis in ML-D sequentially every day, morphological changes of the acrosome during spermiogenesis were successfully recorded. The ML-D is simple in design and useful for culturing testis tissue for inducing and maintaining spermatogenesis with clearer visibility. Due to the new method of sample loading, tissues other than testis should also be applicable.

Neonatal testis growth recreated in vitro by two-dimensional organ spreading.

Organ culture experiments can be hampered by central degeneration or necrosis due to the inadequate permeation of oxygen and nutrients, which deteriorates the function and growth of cultured tissues. In the current study, we aimed to overcome this limitation of organ culture through spreading the tissue two dimensionally on an agarose gel stand and molding into a disc shape by placing a ceiling of polydimethylsiloxane (PDMS) chip, which is highly oxygen permeable. By this, every part of the tissue can receive a sufficient supply of oxygen through PDMS as well as nutrients through the agarose gel below. This method not only prevented central necrosis of tissues, but also supported the tissue growth over time. In addition, such growth, as volume enlargement, could be easily measured. Under these conditions, we examined the effect of several factors on the growth of neonatal mouse testis, and found that follicle stimulating hormone (FSH) and insulin significantly promoted the growth. These results are in good agreement with previous in vivo reports. Notably, the growth achieved over 7 days in our in vitro system is almost comparable to, about 80% of, that observed in vivo. Thus, we successfully monitored the promotion of tissue growth beyond the limits of the conventional organ culture method. This extremely simple method could offer a unique platform to evaluate the growth as well as functional properties of organs, not only the testis but also others as well.

Outcome of flexible ureteroscopy for renal stone with overnight ureteral catheterization: a propensity score-matching analysis.

PURPOSE: To evaluate the influence of overnight ureteral catheterization and determine if routine long-term post-stenting can be avoided in flexible ureterorenoscopy (fURS) procedure for kidney stone. METHODS: Three hundred ninety-three patients who underwent single fURS for kidney stone between January 2013 and June 2016 at a single institute were retrospectively analyzed. The stone-free (SF) and perioperative complication rates in patients with routine long-term post-stenting after fURS (long-term stent group) were compared with those of patients with overnight ureteral catheterization (short-term stent group). Propensity score-matching analysis was used to adjust the difference in baseline preoperative parameters between the two groups. All preoperative parameters were chosen to develop the propensity score, and 74 patients in the short-term stent group were retrospectively matched with the patients in the long-term stent group at a 1:1 ratio. RESULTS: Patient characteristics included age, sex, side of involvement, height, body weight, body mass index, number of stone(s), stone volume, Hounsfield units of stone, preoperative white blood cell count, preoperative C-reactive protein, preoperative creatinine, pretreatment, pre-stenting, stenosis of the ureter, and procedure duration. The SF rates were 91.9 and 93.2% in the short-term and long-term stent groups, respectively. Perioperative complications were 14.9 and 12.2%. No difference was noted between the two groups in terms of SF and perioperative complication rates. CONCLUSIONS: Short-term post-stenting using overnight ureteral catheterization in uncomplicated cases after fURS for kidney stone was as effective as conventional long-term post-stenting in reducing postoperative complications. These preliminary data suggest the possibility that routine long-term post-stenting was unnecessary.

In vitro spermatogenesis: A century-long research journey, still half way around.

BACKGROUND: Spermatogenesis is one of the most complicated cellular differentiation processes in a body. Researchers struggled to find and develop a micro-environmental condition that can support the process in vitro. Such endeavors can be traced back to a century ago and are yet continuing. METHODS: Reports on in vitro spermatogenesis and related works were selected and classified into four categories based on the method used; organ culture, tubule culture, cell culture, and 3-dimensional cell culture methods. Each report was critically reviewed from the present point of view by authors who have been working on in vitro spermatogenesis with organ culture method over a decade. RESULTS: The organ culture method has the longest history and is the most successful method, which produced fertile mouse sperm from spermatogonial stem cells. Formulation of the medium was a key factor, most importantly serum-derived substances. However, factors in the serum that induce and support spermatogenesis in the cultured tissue remain to be identified. In addition, the success of mouse spermatogenesis is yet to be applied to other animals. On looking into the history of cell culture method, it became clear that Sertoli cells as feeder cells play an important role. Even with Sertoli cells, however, spermatogenic development has been limited to small parts of spermatogenesis, a segmented period of meiotic prophase for instance. Recent developments of organoid or 3-dimensional culture techniques are promising but they still need further refinements. CONCLUSION: The study of in vitro spermatogenesis progressed significantly over the last century. We need more work, however, to establish a culture system that can induce and maintain complete spermatogenesis of many if not all mammalian species.

In vitro production of functional sperm in cultured neonatal mouse testes.

Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation. The whole process, therefore, has never been reproduced in vitro in mammals, nor in any other species with a very few exceptions in some particular types of fish. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas-liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.

Spermatogenesis in Explanted Fetal Mouse Testis Tissues.

We previously reported the successful induction of complete spermatogenesis of mice in neonatal testis tissues cultured on agarose gel, with the culture medium supplemented with a bovine serum albumin product, AlbuMAX. This method, however, has not been examined for fetal testis tissues. In this report, we tested the culture method for fetal testes of the Acrosin (Acr)-Gfp transgenic mouse, whose testicular germ cells express GFP from the midmeiotic phase onward, using AlbuMAX-containing medium. The fetal testis, from 19.5 days postcoitum (dpc) back to 14.5 dpc, showed spermatogenic progression and produced haploid cells in culture. On the other hand, testes of 13.5 dpc or earlier did not show the meiotic sign of Acr-Gfp expression. Regardless of the fetal age, tissue masses enlarged during the culture period because of the elongation and thickening of the seminiferous tubules. This simple culture method could be a useful experimental system to investigate fetal testicular development and germ cell biology.

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.

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.

Evaluation and validation of the core lower urinary tract symptom score as an outcome assessment tool for the treatment of benign prostatic hyperplasia: effects of the α1-adrenoreceptor antagonist silodosin.

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.

Generation of a novel germline stem cell line expressing a germline-specific reporter in the mouse.

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh-Venus GS cell line, to our knowledge, is the first one expressing a germ cell-specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation.

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.