The Expression Of PCNA In The Erythroid Cells In Peripheral Blood And Hematogenic Organs Of Di-and Triploidy Rainbow Trout (Oncorhynchus Mykiss)

Proliferating cell nuclear antigen is a kind of protein which only can be found in the nuclei of proliferating cells. It was used as a marker of the proliferation of cells. The located research of the expression of PCNA in peripheral blood and hematogenic organs in rainbow trout could determine the reproductive activity of erythrocytes, and offer basis data for the research of erythrocytes in polyploidy fish.In this paper, the morphology and construction of erythrocytes in peripheral blood of di- and triploid rainbow trout Oncorhychus mykiss(Walbaum)was observed and contrasted. The expression of PCNA in erythrocytes was examined by the method of immunohistochemistry and flow cytometry. And the proportion of the erythrocytes in S phase was detected at the same time. Positioning study was took place in the hematogenic organ to determine the distribution and active degree of the hemopoietic tissues. The results were as follows:1. Contrast with that of diploids, the erythrocytes in triploid rainbow trout was larger and more slender. The volume of the erythrocytes and the nuclei of the erythrocytes in triploid were 1.49 and 1.5 times larger than that of diploids which were corresponded to the expectant theoretical value of 1.5. In peripheral blood of triploid rainbow trout, about 8.5% of erythrocytes nuclei showed abnormal shapes of constriction, dumbbell, and irregular shape. This phenomenon could also be seen in diploids but the proportion was very small which was 0.08%. Immature erythrocytes could also be seen in the peripheral blood of di- and triploid rainbow, and the proportions of which were 8% and 11.9% which had significant difference.2. Brown precipitates could be observed by light microscope in the nuclei of PCNA positive cells after staining by the method of immunohistochemisty. The rates of PCNA positive erythrocytes in the peripheral blood of di- and triploid rainbow were 0.2% and 6.4% in erythroid cells (antibody used in the experiment was anti-PCNA monoclonal antibody (PC10)). The shapes of positive erythrocytes corresponded to immature erythrocyte. The proportions of positive erythrocytes in immature erythrocytes of di- and triploids were 2.5% and 57.9%.3. Determining by flow cytometry, the rate of the erythrocytes in peripheral blood which were under S phase was 0.69% in diploids and 4.05% in triploids and the proportion of antibody labeling cells was 0.37% in diploids and 8.39 in triploids.4. Anti-PCNA monoclonal antibody (PC10) could specifically bind with PCNA in proliferative cells which could be detected by protein electrophoresis which showed a 35-36 kDa protein strap. An immunoreactive polypeptide with an electrophoretic mobility is found in rainbow trout liver, spleen and the peripheral blood cells of triploids. The staining intensity demonstrates that peripheral blood cells of triploids whose cells possess a lower proliferatory capacity.5. TUNEL, which is a method of apoptosis detection was used to determine the abnormal erythrocytes in peripheral blood of di- and triploid rainbow trout. The apoptotic erythroid cells were indicated by the brown reaction products. The result showed that apoptosis erythrocytes could be found both in di- and triploid rainbow trout, and these cells were normal apoptosis erythrocytes. Abnormal erythrocytes showed negative to TUNEL probing, so they were not the cells which undergone a procession of apoptosis. Thus the presumption about abnormal erythrocytes of apoptosis could be eliminated.6. The head kidney of rainbow trout was enriched in hematopoietic tissue but lack of the structures related with excretory system, and the hematopoietic cells proliferated actively which showed that it was one of the most important hematopoietic organs in rainbow trout. In this study, PCNA positive cells in kidney were also centered to the endothelial cells which may suggest that there were interactions between hematopoietic endothelial and renal tubular cells. In spleen strong immunoreactivity is observed, and PCNA is expressed exclusively in the hematopoietic compartment. The positive cells are hematopoietic progenitor cell. A small quantity of hepatic cells was positive staining which dispersed mainly in hepatic sinusoid and the region of subvelamen.