Searching of factors facilitate spermatogonial stem cell proliferation (Investigation with transplantation and in vitro analysis)

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1) Fertilization of recipient mice with donor germ cellsPup mice, aged 8 〜 20 days old, were treated with regional radiation to eliminate innate testis germ cells. Spermatogonial transplantation was performed on those mice. Donor spermatogenesis took place in the host testes pervasively at 1 〜 2 months later. Mating with regular females produced donor germ cell derived progeny. This result indicates that spermatogonial stem cells in the pup testis expanded more rapidly than when they were transplanted in the about recipients.2) GnRH analogue effects on spermatogonial cell proliferationWe analyzed proliferation of spermatogonia using transplantation and confocal laser microscopic analysis. In GnRH analogue treated mice, the number of spermatogonia significantly increased compared to those in control mice, indicating that GnRH analogue enhanced proliferation or suppressed apoptosis of the spermatogonia. Another experiment using S1/S1^d mice as recipients showed that the effect of GnRH analogue is independent from SCF signal.3) Culture of spermatogonial stem cellsTestis cells of GFT mice were cultured for 2 to 3 weeks. To confirm the presence of spermatogonial stem cells, they were transplanted to the testes of pup mouse. Several different culture conditions were tested, including feeder cells (STO, 15P-1), coated dishes with lamina or collagen, madrigal matrix, and so on. Among them cultured cells on the madrigal matrix showed they contained spermatogonial stem cells for such periods.