| In mammalian, Spermatogonial stem cells are the only diploid somatic cells, which can transmit the genetic information to the generations. The culture and differentiation research can support the basis of conservation of mammalian genetic information. It also supports the basis theory to study the spermatogenesis. The purpose of this study was to isolate the spermatogonial stem cell-like cells from murine testicular tissue, which then were induced into haploid germ cells by retinol acid (RA). The spermatogonial stem cell-like cells were purified and enriched by a two-step plating method based on different adherence velocities of spermatogonial stem cells (SSCs) and somatic cells. The cell colonies were presented after cultured in M1-medium for3d. Through alkaline phosphatase (AKP), RT-PCR and indirect immunofluorescence cell analysis, cell colonies were proven to be SSCs. Subsequently, cell colonies of SSCs were cultured in M2-medium containing RA for2d. Then the cell colonies of SSCs were again cultured in M1-medium for6～8d, RT-PCR and indirect immunofluorescence cell analysis were chosen to detect haploid male germ cells. The results are as following:1. SSCs are small, round or oval cells. The high purity of SSCs were obtained by the different adherence velocities, then they were seeded on the Sertoli cells treated with mitomycin C and began division and proliferation after the next day. Using medium containing EGF culture SSCs, the cell colonies emerged in3d and they were clear to see during the5-7d. The cell colonies were radial growth, the cells were small, uniform and oval, whose morphology were the same with SSCs.2. The difference between the Sertoli cells and SSCs was the morphology. SSCs were round and stained bluish violet, while Sertoli cells had an irregular form and were barely stained. The AKP assay together with the images of the cell colonies demonstrated that the colonies of the round cells could be considered as SSCs colonies. Some characteristic genes of germ cells are selected to identify the spermatogonial stem cells and haploid male germ cells. The Ngn3gene is a typical gene of SSCs and the Oct4gene maintain pluripotency and self-renewal. The β-actin gene is a house-keeping gene of SSCs and Sertoli cells. The Integrin alpha6and Integrin beta1genes are considered as the surface markers of SSCs. SSCs expressed the four genes (Oct4, Integrin alpha6, Integrin beta1, Ngn3, and the house keeping gene), while Sertoli cells only expressed the house-keeping gene (β-actin). The RT-PCR analysis indicated different gene expression of SSCs and Sertoli cells. Thus, it was demonstrated that the cell colonies we cultured were SSCs. It is commonly considered that the As (a single) and Apr (a paired) spermatogonia having stem-cell properties. CD9were selected as the surface maker of spermatogonial stem cells. It is clear that the SSCs colonies expressed CD9on their cell surface after culture of3～5d. Although Sertoli cells also expressed the CD9, they showed a small spherical nucleus with a thick rim of cytoplasm. In contrast, SSCs showed a large spherical nucleus with a thin rim of cytoplasm, whose morphology was comparable to mouse SSCs. Thus, it was demonstrated again that the cell colonies we cultured were SSCs.3. The SSC colonies were treated with RA for2d and then cultured in M1-medium for5～8d. The SSCs began to become meiotic and further differentiated into haploid male germ cells after induction by RA leading to the expression of Sycp3and TH2B. The control group for haploid male germ cells was SSCs which did not expressed both genes (not shown). Thus, it was obviously demonstrated that SSCs were induced to haploid male germ cells by RA. Haploid male germ cells largely expressed the protamine1. In contrast, SSCs barely expressed protamine1, demonstrating that SSCs had been differentiated into post-meiotic... |
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