Maturation In Vitro And Vitrification Of Zebrafish Oocytes

Maturation in vitro of zebrafish oocytes promoted with 17a-hydroxyprogesterone were studied with the methods of BCB staining analysis and different concentrations vitrification, different processing time treated with steroid hormone; The condition of vitrification of zebrafish oocytes were screened by different vitrification solution and detection of the survival rate of oocytes after vitrification; the vitrification injury on zebrafish oocytes were studied by detection of the activity of three kinds of mitochondrial ATPase(Ca2+-ATPase ? Mg2+-ATPase and Na+K+-ATPase) and the content of glutathione in the oocytes after vitrification. The main results were as follows:(1) Compared with the experimental results of two methods of BCB staining and measured or observed oocyte diameter and cell morphology to determine the degree of oocyte maturation, the results proved that BCB staining was feasible and convenient. The best dyeing effect of BCB staining was obtained in the concentration of 20 ?mol/L and 150 min staining time.(2) Culture medium without 17?-hydroxyprogesterone was employed as control group, and the medium added with 17?-hydroxyprogesterone in concentrations of 1 nmol/L, 10nmol/L, 100nmol/L and 1000nmol/L as experimental groups, respectively, the zebrafish oocytes were cultured in vitro for 6h, 12 h and 24 h, respectively. The results showed that: 1) 17?-hydroxyprogesterone had no significant effect on the survival rate of zebrafish oocytes; 2) 17?-hydroxyprogesterone in concentrations of 1 nmol / L and 10 nmol / L treatment for 6h, the maturation rates of the oocytes were 50.99 % and 44.30 % respectively, but no significant differences were seen compared with that of the control group, it could be inferred that low concentrations of 17?-hydroxyl progesterone(1 nmol/L and 10 nmol/L) has no obvious promotion of zebrafish oocytes maturation in vitro; 1000 nmol/L 17?-hydroxyprogesterone treatment for 6 h respectively, the maturation rate of oocytes was 3.33 %, The maturation rate decreased significantly compared with the control group, which indicated that high concentrations of 17?-hydroxyl progesterone(1000 nmol/L) had not profit in the oocytes maturation in vitro; Treaded with 100 nmol/L 17?-hydroxyprogesterone for 12 h, the maturation rate(40.72%) was significantly higher than that of the control group(p<0.05), which indicated that it had a significant role in promotion in the oocytes maturation in vitro; 3) The concentrations of 17?-hydroxyprogesterone range of 1 to 1000 nmol/L, treatment for 6 hours, the half mature ratio of zebrafish increased as the concentration increases, and the ratio were higher than that of the control group, and the ratio in the 100 and 1000 nmol/L groups reached 47.92% and 56.48%, respectively, which possessed significant difference compared with the control group(p<0.05), which demonstrated that 17?-hydroxyprogesterone possessed promotion in the oocytes ripe procedure, moreover, the promotion was dose-dependent with the 17?-hydroxyprogesterone concentrations.(3) 9 groups(V1~V9) of vitrification solution were set and the survival rate of the oocytes after vitrification of were detected after 1d, 2d, 4d, 8d, in order to screeing of vitrification of zebrafish oocytes. The results showed that the survival rate of V4 group was the highest. The survival rate of oocytes were 48.89±2.58%?39.35±1.34%?24.18±2.32% and 14.56±1.64% after 1d, 2d, 4d, 8d vitrification, respectively. With the vitrification time increased, the survival rates of oocytes decreased, obviously. The composition of the vitrification solution was: 1.5 mol/L methanol, 6.0 mol/L ethylene glycol and 0.25 mol/L sucrose.(4) The activities of three kinds of mitochondrial ATPase(Ca2+-ATPase ?Mg2+-ATPase and Na+K+-ATPase) and the content of glutathione in the zebrafish oocytes after vitrification were detected. The results showed that: 1)The activities of these three kinds of mitochondrial ATPase of zebrafish oocytes after 1d, 2d, 4d, 8d vitrification were significantly decreased. But the activities of mitochondrial ATPase in group V4 was the highest among all. The activities of Ca2+-ATPase in group V4 were 3.823±0.144U/mgprot ? 3.492±0.084U/mgprot ? 2.554±0.097U/mgprot and1.930±0.076U/mgprot after 1d, 2d, 4d, 8d vitrification, respectively. The activities of Mg2+-ATPase were 4.503±0.144 U/mgprot ? 4.134±0.182U/mgprot ?2.972±0.096U/mgprot and 2.113±0.073 U/mgprot. The activities of Na+K+-ATPase were 7.520±0.198 U/mgprot ?5.828±0.184 U/mgprot ?3.204±0.197 U/mgprot and 2.031±0.176 U/mgprot. 2) The contents of glutathione of zebrafish oocytes after 1d, 2d, 4d, 8d vitrification were significantly decreased. But the content of glutathione of group V4 was the highest. The content of glutathione were 13.37±0.59 mg GSH/gprot ?10.57±0.73 mg GSH/gprot?6.25±0.89 mg GSH/gprot and 4.16±0.57 mg GSH/gprot after 1d, 2d, 4d, 8d vitrification, respectively. 3) All experimental data were analyzed and the results showed that the activities of the oocytes decreased with the increase of vitrification time. It suggested that vitrification could not completely avoid the damages caused by the low temperature and the toxicity of cryoprotectants to zebrafish oocytes.