Identification， Separation and Enrichment of Oogonia from Japanese Flounder (Paralichthys olivaceus)

Abstract: In order to obtain high proportion and plenty of oogonia from Japanese flounder， the differences of isolation of oogonia from the fish of different months of age were compared and analysed by enzyme combination digestion and Percoll gradient centrifugation in this study. In addition， the oogonia were identified by optical observation and immunolocalization of Vasa protein in the germ cells. The results showed that (9.86±1.02)×104 oogonia were obtained from 1 mg of 6-month-old ovaries， which were significantly higher than those of 18 months and 30 months (P<0.05). After Percoll gradient centrifugation， the oogonia were mainly distributed in L-15-20% Percoll gradient fluid and 20%-35% Percoll gradient fluid. The proportions of the oogonia of the two cell zones were more than 75%， which were significantly higher than those of the beginning (P<0.05). Moreover， the cell survival rates were more than 84%， roughly matched those of the pre-purified cells (P>0.05). There were no significantly differences between the number of oogonia before and after purification from 1 mg of 6- or 30-month-old ovaries except for 18-month-old (P>0.05). High proportion and abundant oogonia can be obtained from 6-month old ovary through enzyme combination digestion and Percoll gradient centrifugation， so as to provide the necessary source of cells for further develop germ cell transplantation， gene modification research.

CSTR: 32200.14.cjcb.2018.09.0008