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Spermatogenesis is a complex developmental process of continuous cell division involving a phase of germ cell expansion, meiotic division and cytodifferentiation that ultimately generates mature gametes. The object of this study was the development of optimal culture system in order to induce spermatogenesis in vitro and for its establishment. We collected immature testicular tissues from different developmental-stage of mice and rats which were subsequently cultured on the agarose gel with serum-free culture media. After appropriate culture period, we counted multiple layered seminiferous tubules in the cultured testis tissue to find out whether any differentiation occured following spermatogenesis. Between our experimental mouse strains ICR and C57, as a result, we observed significant increased spermatogenic tubules in case of C57 mouse strain in neonatal stage. In our optimized culture condition, we detected the lectin PNA positive spermatid resulted from spermatogenesis in cultured testis tissue after 6 weeks of incubation. We also injected the lentiviral transduced rat spermatogonial stem cells (SSCs) into recipient testis to generate the transgenic gametes. The testicular fragments were then cultured based on our optimized culture condition. After culturing 8 weeks, we observed expression of GFP in differentiated germ cells including post meiotic germ cells, which suggested the induction of donor-derived spermatogenesis. Also, in order to optimize the in vitro culture system for rat testis, we have tested the additional effect of hormones and growth factors, but could not observe the significant increased effect of hormone and growth factors treated groups than the control. In conclusion, our in vitro tissue culture methods could be applicable for production of in vitro transgenic gametes in various kinds of mammalian species including domestic animal.

Spermatogonial stem cells (SSCs) are adult stem cells that transmit genetic information from the parent to the next generation (progeny) in males, and thus, SSCs have used in germline-modification for generating transgenic animals. In this study, we demonstrated the feasibility of transgenic sperm production by employing an effective busulfan treatment method to prepare recipient pigs for the transplantation of genetically modified donor porcine SSCs. We purified SSCs from pig testis cells by sequentially employing Laminin-coated dishes and culture dishes. The purified cells were transduced with lentivirus expressing enhanced green fluorescent protein (eGFP) at a multiplicity of infection (MOI) of 6 for 9 h. eGFP transduced pig SSCs were then transplanted into the seminiferous tubules of 12 to 16-week-old recipients born to busulfan-treated sows. We obtained six recipient pigs after transplantation and maintained them for more than 6 months. The collected viable spermatozoa from 2 out of 6 recipients were positive for eGFP gene expression in polymerase chain reaction. eGFP-expressing spermatozoa appeared morphologically normal under the microscope. When spermatozoa from these recipients were used for intra cytoplasmic sperm injection, eGFP expression could be detected in the embryos. Furthermore, eGFP colonies were derived from donor-transduced SSCs observed in the recipients’ testes. In summary, we demonstrated the successful production of functional-transgenic spermatozoa by transplantation of porcine SSCs where the transgenic was transduced by employing the lentiviral vector system.

Primordial germ cells (PGCs) as well as fetal germ line stem cells (GSCs) are pluripotent cells. Their differentiation potential is similar to that of embryonic stem cells (ESCs), suggesting that germ line lineage may retain the potential to form pluripotent cells. Here we report successful establishment of multipotent germ line stem cells (mGSCs) from cells obtained from adult mouse testis. We obtained testes from Pou5f1 (Oct4)-GFP transgenic mice and demonstrate that the germ cells present in testes can be converted to pluripotent stem cell-like cells. The newly established mGSCs had similar morphology and growth characteristics as that of ESCs and expressed markers of pluripotency. To assess the differentiation potential of mGSCs in vivo and in vitro, we performed a teratoma formation assay and in vitro differentiation via embryoid body (EB) formation. Multipotent GSC-derived teratomas contained derivatives of all the three embryonic germ layers and differentiated into multiple lineages in vitro, including cardiomyocytes and neural cells. Using real-time qPCR analysis, we compared the gene expression pattern in the newly established mGSCs with those of pluripotent stem cells, germ cells, and testicular cells. The differentiation potential of the newly established mGSCs was comparable to that of ESCs and induced pluripotent stem cells (iPSCs). Multipotent GSCs derived from adult testes can be used for personalized cell-based therapies without the ethical and immunological problems.