Measurement of Glucose 6-Phosphate Dehydrogenase Activity in Bovine Oocytes and Embryos Using Qualitative Fluoresence Microscopy

采用荧光显微镜、光电倍增管和光度计对牛卵巢卵泡卵母细胞和体外受精胚胎的葡萄糖6-磷酸脱氢酶（G6PDH）活性进行了检测。NADPH的荧光响应呈线性，相关系数为0.997，检出限为0.5mM。完整卵母细胞的G6PDH活性不能直接测定。提取液中游离透明带卵泡卵母细胞在LN_2中冷冻，解冻后离心，测定其上清液G6PDH活性。用该方法测定了卵母细胞生发囊、第二中期和两细胞到胚期胚胎的G6PDH活性。胚泡期至四细胞期G6PDH活性为0.67 ~ 0.84 nmol /胚/h，此后活性逐渐降低。桑葚胚期无活性。

The activities of glucose 6-phosphate dehydrogenase (G6PDH) of the follicular oocytes collected from bovine ovaries and the embryos fertilized in vitro were measured using fluorescence microscope with photomultiplier and photometer attachments. The response for the fluorescence of NADPH was found to be linear, with correlation coefficiency of 0.997 and the limit of detection was 0.5mM.The G6PDH activity of intact oocytes could not be measured directly. Then the zona pellucida free follicular oocytes in extraction solution were frozen in LN_2, thawed and centrifuged and the G6PDH activity of their supernatant could be measured. Using this method, the G6PDH activities of oocytesat germinal vesicle and second metaphase stages and embryos at two-cell to morulae stages were determined. The G6PDH activities were 0.67 to 0.84 nmol NADPH generated/embryo/h at germinal vesicle stage to four-cell stage and the activity was decreased thereafter. There was no activity at morulae stage.