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.

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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.

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.

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 ␤ isoform of 14-3-3 proteins promotes tumorigenicity and angiogenesis of rat hepatoma K2 cells. In this study, to analyze the mechanism of 14-3-3␤-induced malignant transformation, yeast two-hybrid screening was performed, and a novel 14-3-3␤-binding factor, FBI1 (fourteen-three-three beta interactant 1), was identified. In vitro binding and co-immunoprecipitation analyses verified specific interaction of 14-3-3␤ 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 FBI1-14-3-3␤ complex could act as a transcriptional silencer, and MKP-1 (MAPK phosphatase-1) was one of the target genes of the FBI1-14-3-3␤ complex. Moreover, chromatin immunoprecipitation analysis demonstrated that FBI1 and 14-3-3␤ 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.