| Objective To establish the approaches of spermatogonia isolation, purification and culture, and explore effect of nickel sulfate on oxidative stress, Caspase3 and Bax mRNA and protein expression in mice spermatogonia in vitro.Methods (1) Healthy Kunming genus male mice of birth 6-8 days by sterilizing with 75% ethanol thoroughly were selected to remove mice testes under sterility condition. Enzymatic digestion (1g/L collagenaseⅣ,1.5g/l hyaluronidase and 0.25% trypsin) combined with Percoll density gradient centrifugation was used to isolate, purificate spermatogonia firstly, and spermatogonia further purificated with adherent culture and identified the spermatogonia purity with staining means of alkaline phosphatase. (2) The spermagonia of logarithmic growth phase administrated with nickel sulfate at the concentration of 50,100,200,400,800μmol/L and the control group treated with culture solution. After exposure 6,12,24 and 36 hours, the spermatigonia were harvested and obtained the supernant by centrifugation after ultrasonication to detect the activities of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-PX), and the content of hydroxyl free radical (-OH(?)) with Microplate Reader. (3) The cultured spermagonia of logarithmic growth phase inoculated to culture flask with volume of each flask 10ml (cells of 1×10′/ml) and administrated with nickel sulfate at the concentration of 200 and 400μmol/L, but the control group treated with culture solution with same volume. After exposure 12,24 hours, the spermatigonia were harvested to detect the mRNA and protein expression of Bax and Caspase3 with Real-time flurescence quantitative PCR and Western blotting techniques.Results (1) After cultured 24 hours, the spermatogonia morphology under inverted microscope showed cells satiation, having glossing and stroung refractive ability, the cells scattered and beading distribution or the chain shape growth, and some cells membrane linked to form cell bridges. There are little cytoplasma and round cell nucleus with 1-3 nucleoli located in nucleus central in spermatogonia. After stained with alkaline phosphatase, the cells cohered by brown precipition were spermatogonia and the percentage of staining positive cells accounted to above 90%. (2) Compared with control group at different exposure concentration group in same exposure time, the content of -OH(?) increased and the activities of GSH-Px and T-AOC inhibited by NiSO4 at dosages of 200,400 and 800μmol/L at exposure 6,12, 24 and 36h respectively (P<0.05), but the content of -OH'increased and the activity of GSH-Px inhibited in NiSO4 50μmol/L group at exposure 36h (P<0.05) and showed obviously dose-effect relationship. Compared with Oh group at different exposure time group in same exposure concentration, the content of -OH'increased and activity of T-AOC inhibited at 6,12,24 and 36h groups in NiSO4 200,400,800μmol/L respectively and the levels of GSH-Px,-OH'and T-AOC had abnomal changes at exposure 24 and 36h groups in NiSO4100μmol/L group respectively (P<0.05), but the levels of -OH'and T-AOC had only abnomal changes at exposure 36h group in NiSO4 50μmol/L (P<0.05), and showed obviously time-effect relationship. (3) After exposure 12h and 24h, compared with control group, the mRNA expression of Bax and Caspase3 were upward in NiSO4 200 and 400μmol/L (P<0.05). Western-blotting results showed the protein expression of Bax and Caspase3 had the ascending tendency gradually with the increase of NiSO4 exposure concentration and exposure time.Conclusion The approaches of enzymatic digestion combined with Percoll density gradient centrifugation and adhent culture of spermatogonia could be feasible and acquire higher purity spermatogonia in mice in vitro. The results suggested that the damage of mice spermatogonia induced by nickel sulfate could be related to the enhancement of oxidative stress, and mRNA and protein expression upward of Bax and Caspase-3. |
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