| To investigate the condition and method for the in vitro proliferation of spermatogonial stem cells (SSCs). Germ cells were isolated from testes of KM mice aged 6-8 days, and were enriched using Percoll grads centrifugation; sertoli cells treated with mitomycin C were used as feeder cells, the culture medium was DMEM supplemented with 5% FBS, 10~3 U/ml leukemia inhibitory factor (LIF). To confirm whether the cultured germ cells were still stem cells, immunofluorescence staining using Thy-1 as surface marker was performed. On the feeder layer, the anchoring time of SSCs was 6 h-9 h; SSCs began to proliferate 48 h later, and rapid proliferation appeared on the 12th day. On the 20th day, the number of SSCs was 45-245 times than that of subculturing, and the cluster of cells was 26±4 per field under 100×microscope. According to the results of immunofluorescence staining, the cultured cells were stem cells. But after 20 days on culture, the germ cells were seemingly dying. The culture condition is suitable for proliferation of SSCs in vitroTo investigate whether estrogen stimulates SSCs self-renew in cryptorchid mice. 10-day mice rendered experimental cryptorchidism, then treated subcutaneously with different doses of 17p-estradiol (E2) once a day. 35 days later, histological analysis and immunofluorescence were performed. On the other hand, serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured. Resultsshowed that low dose of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of SSCs.Testes in the high-dose-estradiol-treated mice were used for proteomics analysis, and the total protein in testis was extracted. And the differently expressed proteins between estradiol-treated cryptorchid mice and control groups were screened, and compared by the proteomic techniques. In detail, through the separation of the two-dimensional gel electrophoresis (2-DE), twenty differential protein spots were found, and nine of them digested and identified by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum. And among them four proteins were identified, as phosphatidylethanolamine-binding proteini (PEBP1), HERP (HES-related basic helix-loop-helix protein), Stathmin (STMN 1) and one unnamed protein product (Px). HERP expressed as follows: E2-treated mice>olive oil-treated mice>sexually mature mice. But the other three proteins the other way round.To investigate the function of these differential proteins in testis, we cloned the gene of Stathmin and fused it with EGFP gene, and transfected pEGFP-stathmin into sertoli and SSCs. The results showed that stathmin in SSCs were transported from cytoplasm into nucleus, but not in sertoli. Cell number counting within one week after transfection suggested that stathmin could stimulate germ cells proliferation. The localization of stathmin in testis was investigated using in situ hybridization histochemistry. The result indicated that the mRNA of stathmin express highly in spermatogonia, and dimly in the primary spermatocytes and round spermatids, but no in other type of germ cells. All these suggest that stathmin is involved in proliferation of SSCs, perhaps proliferation towards differentiation...
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