Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Animal Reproduction  

DOI： 10.1262/jrd.2023-093

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Mammalian spermatogenesis is a heat-vulnerable process that occurs at low temperatures, and elevated testicular temperatures cause male infertility. However, the current reliance on in vivo assays limits their potential to detail temperature dependence and destructive processes. Using ex vivo cultures of mouse testis explants at different controlled temperatures, we found that spermatogenesis failed at multiple steps, showing sharp temperature dependencies. At 38 °C (body core temperature), meiotic prophase I is damaged, showing increased DNA double-strand breaks (DSBs) and compromised DSB repair. Such damaged spermatocytes cause asynapsis between homologous chromosomes and are eliminated by apoptosis at the meiotic checkpoint. At 37 °C, some spermatocytes survive to the late pachytene stage, retaining high levels of unrepaired DSBs but do not complete meiosis with compromised crossover formation. These findings provide insight into the mechanisms and significance of heat vulnerability in mammalian spermatogenesis.

DOI： 10.1038/s42003-022-03449-y

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Other Link： https://www.nature.com/articles/s42003-022-03449-y

Publishing type：Research paper (scientific journal)  

© 2021, The Author(s). In vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

DOI： 10.1038/s41598-021-82792-2

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Language：English   Publishing type：Research paper (scientific journal)  

Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.

DOI： 10.1016/j.stem.2021.08.005

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

<jats:title>Abstract</jats:title>
<jats:p>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.</jats:p>

DOI： 10.1093/biolre/ioab104

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Language：English   Publishing type：Research paper (scientific journal)  

Spatially fractionated radiation therapy (SFRT) has been based on the delivery of a single high-dose fraction to a large treatment area that has been divided into several smaller fields, reducing the overall toxicity and adverse effects. Complementary microbeam studies have also shown an effective tissue-sparing effect (TSE) in various tissue types and species after spatially fractionated irradiation at the microscale level; however, the underlying biological mechanism remains elusive. In the current study, using the combination of an ex vivo mouse spermatogenesis model and high-precision X-ray microbeams, we revealed the significant TSE for maintaining spermatogenesis after spatially fractionated microbeam irradiation. We used the following ratios of the irradiated to nonirradiated areas: 50:50, 150:50 and 350:50 µm-slit, where approximately 50, 75 and 87.5% of the sample was irradiated (using center-to-center distances of 100, 200 and 400 µm, respectively). We found that the 50 and 75% micro-slit irradiated testicular tissues showed an almost unadulterated TSE for spermatogenesis, whereas the 87.5% micro-slit irradiated tissues showed an incomplete TSE. This suggests that the TSE efficiency for spermatogenesis is dependent on the size of the nonirradiated spermatogonial stem cell pool in the irradiated testicular tissues. In addition, there would be a spatiotemporal limitation of stem cell migration/competition, resulting in the insufficient TSE for 87.5% micro-slit irradiated tissues. These stem cell characteristics are essential for the accurate prediction of tissue-level responses during or after SFRT, indicating the clinical potential for achieving better outcomes while preventing adverse effects.

DOI： 10.1667/RADE-19-00018.1

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Language：English   Publishing type：Research paper (scientific journal)  

In vitro spermatogenesis, which produces fertile spermatozoa, has been successfully performed using an organ culture method from murine tissue. Here, we provide a dataset of time-course microarray transcriptome data of in vitro cultured neonate murine testes and age-matched in vivo-derived testes. The dataset presented here is related to the article titled "Transcriptome analysis reveals inadequate spermatogenesis and immediate radical immune reactions during organ culture in vitro spermatogenesis" published in Biochemical and Biophysical Research Communications in 2020 [1]. The raw data and pre-processed data are publicly available on the GEO repository (accession number GSE147982). Furthermore, the dataset provided here includes additional metadata, detailed explanations of the experiment, results of pre-processing, analysis scripts, and lists of differentially expressed genes from in vitro culture testes and in vivo testes at each time point.

DOI： 10.1016/j.dib.2020.106482

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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.

DOI： 10.1016/j.bbrc.2020.06.161

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© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology 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.

DOI： 10.1096/fj.202000245R

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Radiotherapy can result in temporary or permanent gonadal toxicity in male cancer patients despite the high precision and accuracy of modern radiation treatment techniques. Previous radiobiological studies have shown an effective tissue-sparing response in various tissue types and species following exposure to spatially fractionated radiation. In the present study, we used an ex vivo mouse testicular tissue culture model and a conventional X-ray irradiation device to evaluate the tissue-sparing effect (TSE) of spatially fractionated X-rays for the protection of male fertility from radiotherapy-related adverse effects. We revealed a significant TSE for maintaining spermatogenesis in the ex vivo testes model following spatially fractionated X-ray irradiation. Moreover, we experimentally propose a possible mechanism by which the migration of spermatogonial cells, from the non-irradiated areas to the irradiated ones, in irradiated testicular tissue, is essential for the TSE and maintaining spermatogenesis. Therefore, our findings demonstrate that the control of TSE following spatially fractionated X-rays in the testes has a considerable potential for clinical application. Interdisciplinary research will be essential for further expanding the applicability of this method as an approach for the preservation of male fertility during or after radiotherapy.

DOI： 10.3390/jcm9041089

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>
Microbeam radiotherapy (MRT) is based on a spatial fractionation of synchrotron X-ray microbeams at the microscale level. Although the tissue-sparing effect (TSE) in response to non-uniform radiation fields was recognized more than one century ago, the TSE of MRT in the testes and its clinical importance for preventing male fertility remain to be determined. In this study, using the combination of MRT techniques and a unique <italic>ex vivo</italic> testes organ culture, we show, for the first time, the MRT-mediated TSE for the preservation of spermatogenesis. Furthermore, our high-precision microbeam analysis revealed that the survival and potential migration steps of the non-irradiated germ stem cells in the irradiated testes tissue would be needed for the effective TSE for spermatogenesis. Our findings indicated the distribution of dose irradiated in the testes at the microscale level is of clinical importance for delivering high doses of radiation to the tumor, while still preserving male fertility.

DOI： 10.1038/s41598-019-48772-3

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Other Link： http://www.nature.com/articles/s41598-019-48772-3

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/rmb2.12291

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer New York  

DOI： 10.1007/978-1-4939-8831-0_5

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/bit.26822

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

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.

DOI： 10.1016/j.bbrc.2018.04.180

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Radiation Research Society  

The formation of sperm by the testes through the process of spermatogenesis is highly radiosensitive and can be affected by environmental, occupational and therapeutic radiation exposures. In this study, we applied an ex vivo mouse testis organ culture as an experimental model of spermatogenesis to investigate the radiobiological effects and to demonstrate its feasibility as a tool to determine response to complex, modulated radiation fields. This model uses Acr-GFP transgenic mice, which express the marker green fluorescent proteins specific for meiosis to allow observation of functional changes in real-time that can be used to analyze radiation-induced changes in the process of spermatogenesis. Our results showed that the model can accurately reproduce radiation-induced male germ cell toxicity, such as temporary infertility and permanent sterility. Furthermore, using a monochromatic X-ray microbeam, we applied this model to investigate the effects of heterogeneous radiation fields on testis tissue ex vivo. Our model represents a unique application in the field, which offers significant potential for gaining further mechanistic insight into radiation effects on the process of spermatogenesis.

DOI： 10.1667/RR14957.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

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.

DOI： 10.1371/journal.pone.0192884

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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.

DOI： 10.1038/s41598-017-15799-3

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A search for early response genes that are activated following germ cell induction from mouse embryonic stem cells in vitro led us to the isolation of a long noncoding RNA that contains a SINE (short interspersed element)-B1F motif that was named R53. In situ hybridization and northern blot analyses revealed that the R53 subfragment RNA bears a B1F motif, is processed from the primary transcript, is expressed in adult testis and is predominantly localized in meiotic metaphase chromatin during spermatogenesis. Recent studies of chromosome-associated RNAs have explored novel functions of noncoding RNAs. Specifically, chromosome-bound noncoding RNAs function not only as structural components of chromosome but also as scaffolds that recruit epigenetic modulators for transcriptional regulation, and they are dynamically rearranged during the cell cycle. However, few studies have explored meiotic chromatin; thus, R53 RNA appears to be the first long noncoding RNA to be tightly associated with the metaphase chromatin during spermatogenesis. Furthermore, R53 knockdown using a lentivirus-mediated RNAi injected into mouse testis and organ culture of the fragments revealed a remarkable reduction in postmeiotic cells and irregular up-regulation of several postmeiotic genes, which suggests the possibility that the SINE-B1-derived noncoding RNA R53 plays an indispensable role in the transcriptional regulation of key spermatogenesis genes.

DOI： 10.1371/journal.pone.0179585

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background: The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments. Methods: Testes from postnatal day 5 B6: CBA-Tg(Acrv1-EGFP) 2727Redd/J male mice were cultured in knockout serum replacement (KSR) or Albumax I (Albumax) medium. Enhanced green fluorescent protein (EGFP) expression was examined on days 35, 42, 45, and 49 of culture. Histology and flow cytometry were performed for testis morphology and spermatid differentiation. Results: EGFP signals were first observed in round spermatids on day 22 of culture (corresponding to postnatal day 27) and were observed until the end of culture, indicating testis-specific protein expression. A-kinase anchor protein 4 expression, a marker of elongated spermatid (step 15-16) occurred earlier in explants cultured in KSR than Albumax medium (typically day 35 and after day 42 of culture, respectively). The percentage of seminiferous tubules with elongated spermatid was higher in Albumax than KSR medium from days 45 to 49 of culture. Conclusion: Albumax medium may facilitate or support better morphology and spermatid production than KSR medium. Further studies need to improve spermatid production and refinement of this in vitro testis culture system that may be useful as a supplement to current male reproductive toxicity testing or an alternative model in cases where in vivo testing may be unfeasible. (C) 2017 Wiley Periodicals, Inc.

DOI： 10.1002/bdr2.1002

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFR alpha 1)positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/beta-catenin signaling promotes spermatogonial differentiation and reduces the GFR alpha 1(+) cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRa1(+) cells and confers resistance to the Wnt/b-catenin signaling. Shisa6(+) cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury)(+) cells that are found largely overlapped with Shisa6(+) cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.

DOI： 10.1016/j.stemcr.2017.01.006

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

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 Albu-MAX-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.

DOI： 10.1095/biolreprod.116.140277

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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.

DOI： 10.1038/srep21472

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Other Link： http://orcid.org/0000-0002-6198-2234

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

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-targetedGS 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.

DOI： 10.1016/j.stemcr.2015.05.011

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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/Sl(d) 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.

DOI： 10.1371/journal.pone.0130171

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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.

DOI： 10.1038/ncomms5320

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

The Drosophila piwi gene has multiple functions in soma and germ cells. An in vitro system provides a powerful tool for elucidating PIWI function in each cell type using stable cell lines originating from germline stem cells (GSCs) and ovarian soma of adult ovaries. We have described methods for the maintenance and expansion of GSCs in an established cell line (fGS/OSS) and an in situ hybridization method for analyzing piwi. © 2014 Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-62703-694-8_2

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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.

DOI： 10.1038/nprot.2013.138

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

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.

DOI： 10.1095/biolreprod.113.108613

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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. genesis 51:498-505. (c) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/dvg.22391

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English  

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.

DOI： 10.1007/978-1-62703-38-0_41

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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.

DOI： 10.1073/pnas.1211845109

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Other Link： http://orcid.org/0000-0002-6198-2234

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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.

DOI： 10.1038/ncomms1478

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publisher：SOC STUDY REPRODUCTION  

DOI： 10.1095/biolreprod.110.089342

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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(1,2). The whole process, therefore, has never been reproduced in vitro in mammals(3-5), nor in any other species with a very few exceptions in some particular types of fish(6,7). 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.

DOI： 10.1038/nature09850

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

It is now possible to make mouse spermatogonial stem cells (SSCs) proliferate in vitro. However, these cultured cells, called germ-line stem (GS) cells, consist of not only SSCs but also a greater number of progenitor spermatogonia. Moreover, isolated GS cells barely proliferate. To elucidate the nature of SSCs and progenitor spermatogonia, we adapted a microdrop culture system to GS cells. Using a micromanipulator, individual microdrops were seeded with clusters or dissociated known numbers of GS cells. The number of surviving colonies was determined after 30 days. The proliferation rate of GS cells in microdrops increased as the number of GS cells seeded increased. It was observed that as few as three GS cells seeded in a microdrop can proliferate and expand the colony size. Those GS cells of expanded colonies were able to proliferate following subculture and underwent spermatogenesis in the host testis after transplantation into the seminiferous tubules of recipient mice. These data revealed that SSCs can multiply in a microdrop culture system. Microdrop culture offers a novel tool to elucidate the nature of SSCs in regard to their self-renewing capacity and can serve as a monitoring system of culture conditions for the self-renewal of SSCs.

DOI： 10.1095/biolreprod.109.082800

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

The germline is segregated from the remainder of the soma during early embryonic development in metazoan species. In Drosophila, female primordial germ cells (PGCs) continue to proliferate during larval development, and become germline stem cells at the early pupal stage. To elucidate the roles of growth factors in larval PGC division, we examined expression patterns of a bone morphogenetic protein (BMP) growth factor, Decapentaplegic (Dpp), and Hedgehog (Hh), along with factors downstream of each, in the ovary during larval development. Dpp signaling appeared in the ovarian soma from early larval development, and was prominent in the terminal filament cells at late larval stage, whereas Hh appeared in the ovarian soma and PGCs from the third instar larval stage. The number of PGCs decreased when components of these signal transduction pathways were abrogated by RNAi in the PGCs, indicating that both Dpp and Hh signals directly regulate PGC proliferation. Experiments on the up-and down-regulation of Dpp and Hh with a tissue-specific Gal4 driver indicated that Dpp and Hh act as extrinsic and autocrine growth factors. Furthermore, heat-pulse experiments with hs-Gal4 showed that Dpp is active in PGC proliferation throughout larval development, whereas Hh has effects only during late larval development. In addition to Dpp, the reduction of Glass bottom boat (Gbb), another BMP molecule, caused a decrease in the number of PGCs and initiation of larval PGCs differentiation into cystocytes, indicating that Gbb functions to promote PGC division and repress differentiation.

DOI： 10.2108/zsj.27.804

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Achieving mammalian spermatogenesis in vitro has a long history of research but remains elusive. The organ culture method has advantages over the cell culture method, because germ cells are in situ albeit the tissue as a whole is in vitro. The method was used in the 1960s and 1970s but encountered difficulties in inducing complete meiosis, i.e., in getting meiosis to proceed beyond the pachytene stage. In the present study, we reevaluated the organ culture method using two lines of transgenic mice, Acr-GFP and Gsg2 (haspin)-GFP mice, whose germ cells express green fluorescent protein (GFP) at the mid and end stages of meiosis onward, respectively. Immature testicular tissues from these mice, ranging from 4.5 to 14.5 days postpartum, were cultured on the surface of the medium, providing a liquid-gas interface. Culturing testicular tissues of all ages tested resulted in the expression of both Acr-and Gsg2-GFP. Round spermatids were identified by a combination of Gsg2-GFP expression, cell size, and the presence of a single nucleus with a dot stained by Hoechst. In addition, the chromosome number of one of such presumptive spermatids was found to be 20 by the premature chromosome condensation method. As our semiquantitative assay system using GFP expression grading was useful for monitoring the effects of different environmental factors, including temperature, oxygen concentration, and antiretinoic molecules, further improvement of the culture conditions should be possible in the future.

DOI： 10.1095/biolreprod.110.083899

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Other Link： http://orcid.org/0000-0002-6198-2234

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The germline cells of Drosophila are derived from pole cells, which form at the posterior pole of the blastoderm and become primordial germ cells (PGCs). To elucidate the signal transduction pathways for the development of embryonic PGCs, we examined the effects of various growth factors on the proliferation of PGCs. Up- and down-regulation of Wingless (Wg) in both of soma and PGCs caused an increase and a decrease in the number of PGCs, respectively. The Wg/beta-catenin signaling pathway began to occur in PGCs at the same time as the PGCs began to divide during the embryonic stage in both sexes. In addition, PGCs were found to produce wg mRNA as they begin to divide. Thus, Wg functions as an autocrine factor to initiate mitosis in embryonic PGCs. Decapentaplegic affected the growth of PGCs from the end of the embryonic stage. The results indicate that these growth factors regulate the division of embryonic PGCs in a stage-specific manner. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mod.2008.01.004

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Other Link： http://orcid.org/0000-0002-6198-2234

DOI： 10.11477/mf.1409208497

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Language：Japanese   Publisher：(一社)日本生殖医学会  

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Language：Japanese   Publisher：学研メディカル秀潤社  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2011302606

Event date： 2018.12

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Event date： 2016.8

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Event date： 2013.9

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Event date： 2011

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Event date： 2010

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Presentation type：Oral presentation (invited, special)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (invited, special)  

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Presentation type：Oral presentation (invited, special)  

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