Generation of rat offspring using spermatids produced through in vitro spermatogenesis.

An in vitro spermatogenesis method using mouse testicular tissue to produce fertile sperm was established more than a decade ago. Although this culture method has generally not been effective in other animal species, we recently succeeded in improving the culture condition to induce spermatogenesis of rats up to the round spermatid stage. In the present study, we introduced acrosin-EGFP transgenic rats in order to clearly monitor the production of haploid cells during spermatogenesis in vitro. In addition, a metabolomic analysis of the culture media during cultivation revealed the metabolic dynamics of the testis tissue. By modifying the culture media based on these results, we were able to induce rat spermatogenesis repeatedly up to haploid cell production, including the formation of elongating spermatids, which was confirmed histologically and immunohistochemically. Finally, we performed a microinsemination experiment with in vitro produced spermatids, which resulted in the production of healthy and fertile offspring. This is the first demonstration of the in vitro production of functional haploid cells that yielded offspring in animals other than mice. These results are expected to provide a basis for the development of an in vitro spermatogenesis system applicable to many other mammals.

Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control.

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.

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.

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.

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 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.

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

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

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

With the increasing cure rate of paediatric cancers, infertility, as one of the adverse effects of treatments, has become an important concern for patients and their families. Since semen cryopreservation is applicable only for post-pubertal patients, alternative pre-pubertal measures are necessary. Here we demonstrate that testis tissue cryopreservation is a realistic measure for preserving the fertility of an individual. Testis tissues of neonatal mice were cryopreserved either by slow freezing or by vitrification. After thawing, they were cultured on agarose gel and showed spermatogenesis up to sperm formation. Microinsemination was performed with round spermatids and sperm, leading to eight offspring in total. They grew healthily and produced progeny upon natural mating between them. This strategy, the cryopreservation of testis tissues followed by in vitro spermatogenesis, is promising to preserve the fertility of male paediatric cancer patients in the future.

In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method.

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.

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

It is known that cells of testis tissues in fetal or neonatal periods have the ability to reconstruct the testicular architecture even after dissociation into single cells. This ability, however, has not been demonstrated effectively in vitro. In our present study, we succeeded in reconstructing seminiferous tubules in vitro which supported spermatogenesis to meiotic phase. Testis cells of neonatal mice were dissociated enzymatically into single cells. The cells formed aggregates in suspension culture and were transferred to the surface of agarose gel to continue the culture with a gas-liquid interphase method, where a tubular architecture gradually developed during the following 2 weeks. Immunohistological examination confirmed Sertoli cells forming tubules and germ cells inside. With testis tissues of Acr-GFP transgenic mice, whose germ cells express GFP during meiosis, cell aggregates formed a tubular structure and showed GFP expressions in their reconstructed tissues. Meiotic figures were also confirmed by regular histology and immunohistochemistry. In addition, we mixed cell lines of spermagonial stem cells (GS cells) into the testis cell suspension, and found the incorporation of GS cells in the tubules in reconstructed tissues. When GS cells derived from Acr-GFP transgenic mice were used, GFP expression was observed, indicating that the spermatogenesis of GS cells was proceeding up to the meiotic phase. This in vitro reconstruction technique will be a useful method for the study of testis organogenesis and spermatogenesis.

In vitro spermatogenesis using an organ culture technique.

Research on in vitro spermatogenesis has a long history and remained to be an unaccomplished task until very recently. In 2010, we succeeded in producing murine sperm from primitive spermatogonia using an organ culture method. The fertility of the sperm or haploid spermatids was demonstrated by microinsemination. This organ culture technique uses the classical air-liquid interphase method and is based on conditions extensively examined by Steinbergers in 1960s. Among adaptations in the new culture system, application of serum-free media was the most important. The system is very simple and easy to follow.

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

Male infertility is most commonly caused by spermatogenic defects or insufficiencies, the majority of which are as yet cureless. Recently, we succeeded in cultivating mouse testicular tissues for producing fertile sperm from spermatogonial stem cells. Here, we show that one of the most severe types of spermatogenic defect mutant can be treated by the culture method without any genetic manipulations. The Sl/Sl(d) mouse is used as a model of such male infertility. The testis of the Sl/Sl(d) mouse has only primitive spermatogonia as germ cells, lacking any sign of spermatogenesis owing to mutations of the c-kit ligand (KITL) gene that cause the loss of membrane-bound-type KITL from the surface of Sertoli cells. To compensate for the deficit, we cultured testis tissues of Sl/Sl(d) mice with a medium containing recombinant KITL and found that it induced the differentiation of spermatogonia up to the end of meiosis. We further discovered that colony stimulating factor-1 (CSF-1) enhances the effect of KITL and promotes spermatogenesis up to the production of sperm. Microinsemination of haploid cells resulted in delivery of healthy offspring. This study demonstrated that spermatogenic impairments can be treated in vitro with the supplementation of certain factors or substances that are insufficient in the original testes.

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

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

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.

Proliferation of mouse spermatogonial stem cells in microdrop culture.

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.

In vitro murine spermatogenesis in an organ culture system.

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.

A novel binding factor of 14-3-3beta functions as a transcriptional repressor and promotes anchorage-independent growth, tumorigenicity, and metastasis.

The 14-3-3 proteins form a highly conserved family of dimeric proteins that interact with various signal transduction proteins and regulate cell cycle, apoptosis, stress response, and malignant transformation. We previously demonstrated that the beta isoform of 14-3-3 proteins promotes tumorigenicity and angiogenesis of rat hepatoma K2 cells. In this study, to analyze the mechanism of 14-3-3beta-induced malignant transformation, yeast two-hybrid screening was performed, and a novel 14-3-3beta-binding factor, FBI1 (fourteen-three-three beta interactant 1), was identified. In vitro binding and co-immunoprecipitation analyses verified specific interaction of 14-3-3beta with FBI1. The strong expression of FBI1 was observed in several tumor cell lines but not in non-tumor cell lines. Forced expression of antisense FBI1 in K2 cells inhibited anchorage-independent growth but had no significant effect on cell proliferation in monolayer culture. Down-regulation of FBI1 also inhibited tumorigenicity and metastasis accompanying a decrease in MMP-9 (matrix metalloproteinase-9) expression. In addition, the duration of ERK1/2 activation was curtailed in antisense FBI1-expressing K2 cells. A luciferase reporter assay revealed that the FBI1-14-3-3beta complex could act as a transcriptional silencer, and MKP-1 (MAPK phosphatase-1) was one of the target genes of the FBI1-14-3-3beta complex. Moreover, chromatin immunoprecipitation analysis demonstrated that FBI1 and 14-3-3beta were presented on the MKP-1 promoter. These results indicate that FBI1 promotes sustained ERK1/2 activation through repression of MKP-1 transcription, resulting in promotion of tumorigenicity and metastasis.