Studies On Isolation,Purification,Cryopreservation,Culture,Transplantation Of Mouse Spermatogonial Stem Cells And Transgenic Mouse Mediated By Spermatogonial Stem Cells In Vivo

Spermatogonial stem cells (SSCs) self-renew can maintain the number of themselves and can produce daughter cells that commit to differentiate into spermatozoa throughout adult life of the male. Up to date, SSCs is a focus in the field of developmental biology of animal spermatozoa, which has widely prospects of application in clinical of veterinarian and producing transgenic animals. In this study, mouse SSCs were used for cryopreservation system, culture system and transgenic mouse mediated by mouse SSCs in vivo, in order to prepare the ground for the establishment of SSCs lines and offer cell model for growth and differentiation of cells. The specific test results were as follows:1. The germ cell suspension was obtained by two-step enzymatic digestion from KM mouse at 6-day-old. SSCs were isolated and purified using Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish. Cells were cultured in MEMαmedium supplemented with 10% fetal bovine serum (FBS) at 37℃, in a humidified atmosphere with 5%CO₂. Growth and morphologic changes of cells were observed. Result: seminiferous tubules of 6-day-old mouse were mainly composed of spermatogonial stem cells and Sertoli cells. The purity of the SSCs was up to 80%. SSCs mainly distributed in 28%～36% Percoll gradient.2. Mouse SSCs were isolated from KM mouse at 6-day-old using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen used different freezing medium and cooling rate. After thaw, cells were cultured in minimum essential medium alpha(MEMα)supplemented with 10% fetal calf serum(FCS),100 ng/mL Glial cell line-derived neurotrophic factor (GDNF) . SSCs survived as well as proliferation were examined by WST-8 colorimetric assay. To confirm whether the cultured 96h germ cells were still stem cells, alkaline phosphatase and Reverse Transcription- Polymerase Chain Reaction (RT-PCR) was performed, transplantation of SSCs into recipient mice by testis rete injection. Results demonstrate that the best method to cryopreserve SSCs is freezing medium with 10% dimethyl sulfoxide (DMSO), 10% FCS, 0.07mol/L sucrose and 1℃/min cooling rate, the viability of cell is more than 84%. Although noncontrolled-rate freezing protocol leads to a little lower cell viability than that of controlled-rate freezing protocol, it is a simple and effective cropreservation method for mouse SSCs. What is more, the anchoring time of SSCs in this method was 8h～12h after thaw, SSCs began to proliferate 24h later, and rapid proliferation appeared on the 48h, colonies composed by 20～25 cells in 96h, when SSCs had proliferated nearly 5 times; Transplantation of SSCs after thaw into recipient mice can result in donorderived spermatogenesis. In a word, the cryopreservation condition we used could reserve SSCs' characteristic; culture condition is suitable for proliferation of frozen-thawed SSCs.3. To study the feasibility of establishing transgenic mice carrying enhanced green fluorescent protein gene by means of testis rete injection. Plasmid pEGFP-N1 packed by liposome was injected into the testis rete of 4 weeks male KM mice using micropipette, which were made to mate with spontaneous oestrus female mice at least 6 weeks after the injection, Genomic DNA was extracted from the offspring of the founders of the found mice for Polymerase chain reaction and DNA dot blotting analysis. Among the 4 mice receiving the microinjection, 3 survived and retained their mating ability and fertility, and among their 63 offspring 2 were positive for EGFP DNA as demonstrated by PCR and DNA dot blotting analysis. It is possible to use testis rete injection to establish transgenic animals.