Mitsuru Komeya

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.

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/Sld mouse is used as a model of such male infertility. The testis of the Sl/Sld 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/Sld 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.

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.

In Vitro Reconstruction of Mouse Seminiferous Tubules Supporting Germ Cell Differentiation

ABSTRACT Cells of testicular tissues during 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 the present study, we reconstructed seminiferous tubules in vitro that supported spermatogenesis to the meiotic phase. First, testicular cells of neonatal mice were dissociated enzymatically into single cells. Then, 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, and a tubular architecture gradually developed over the following 2 wk. Immunohistological examination confirmed Sertoli cells forming tubules and germ cells inside. With testicular tissues of Acr-GFP transgenic mice, the germ cells of which express GFP during meiosis, cell aggregates formed a tubular structure and showed GFP expression in their reconstructed tissues. Meiotic figures were also confirmed by regular histology and immunohistochemistry. In addition, we mixed cell lines of spermatogonial stem cells (GS cells) into the testicular cell suspension and found the incorporation of GS cells in the tubules of 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 testicular organogenesis and spermatogenesis.

In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues

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.

Spermatogonial stem cells: Progress and prospects

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.

Pumpless microfluidic system driven by hydrostatic pressure induces and maintains mouse spermatogenesis in vitro

Three-dimensional aggregation and organ culture methods are critical for recreating in vivo cellular phenomena outside the body. Previously, we used the conventional gas liquid interphase organ culture method to induce complete mouse spermatogenesis. After incorporating microfluidic systems, we achieved a significant increase in efficiency and duration of spermatogenesis. One of the major drawbacks preventing the popularization of microfluidics, however, is the use of a power-pump to generate medium flow. In this study, we produced a pumpless microfluidic device using hydrostatic pressure and a resistance circuit to facilitate slow, longer lasting medium flow. During three months of culture, results in induction and maintenance of spermatogenesis showed no difference between pumpless and pump-driven devices. Correspondingly, the spermatogonial population was favorably maintained in the pumpless device compared to the conventional method. These results show the advantage of using microfluidic systems for organ culture experiments. Our pumpless device could be applied to a variety of other tissues and organs, and may revolutionize organ culture methods as a whole.

In vitro mouse spermatogenesis with an organ culture method in chemically defined medium

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

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.

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

PurposeTo 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.MethodsThree 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.ResultsPatient 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.ConclusionsShort-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.