Study On Gonadal Apoptosis Signal Pathway During Natural Sex Reversal In Monopterus Albus

Ricefield eel,Monopterus albus,is a protogynous hermaphrodite fish.Apoptosis plays an important role in the disappearance of oocytes.In order to explore the apoptotic mechanism of oocytes during the sex reversal of ricefield eel,in the present sduty,the apoptotic rate in different developmental stage gonads was examined by flow cytometry,the expression pattern of genes and proteins associated with apoptotic signaling pathways were analyzed by real-time quantity PCR and enzyme-linked-immunosorbent serologic assay and immunohistochemistry.Furthermore,the gene expression related apoptosis was also detected in the ovary,treated with H₂O₂ and Z-VAD-FMK.The main results are as follows:1.The results of apoptotic rate of gonads showed apoptotic signals can be detected in all developmental stages gonads by flow cytometry.The apoptotic rate significantly increased at the early intersexual stage firstly（P<0.05）,and then significantly decreased during the sexual reversal.However,there was no significant difference in the apoptotic rate of ovary and testis（P>0.05）.The relative content of apoptosis related proteins was detected using the enzyme linked immunosorbent assay kit.These results showed that Cyt-C of the mitochondrial pathway,Caspase-8 of the death receptor pathway and Caspase-12 of the endoplasmic reticulum pathway were expressed in all developmental stage gonad,and the relative content of Cyt-C was significantly higher than that of Caspase-12 and Caspase-8（P<0.05）.The Cyt-C content increased at the early intersexual stage ovary（P<0.05）,and then decreased with the development of sex reversal.The genes of three apoptotic signaling pathways were expressed in all developmental stages of gonads.In total,the expression level of bax of the mitochondrial pathway is higher than tnfr1 of the death receptor pathway and calpain of the endoplasmic reticulum pathway.These results suggest that mitochondrial apoptotic pathway may play an important role in the process of natural sex reversal in Monopterus albus.2.The results of Western blotting showed that the expected target band（12 k Da）of Cyt-C was detected in the gonad tissue.The positival signals were localized in the metaphase gland of yolk oocyte and mid-to late-vitellogenic stage oocyte by immunohistochemistry.The signals in primary oocyte were stronger than that in cortical alveoli oocyte in the ovary.In intersexual phase gonads,the positive signals were in the cytoplasm of oocytes.These suggests that Cyt-C may involv in the apoptotic oocytes of Monopterus albus.3.The results of in vitro incubation showed that the expression of apoptotic genes were all up-regulation in ovary by treated with the different concentrations of H₂O₂.The expression of apaf1 was highest in each treated group at 2h,however,the expression of bax reached the highest at 4h and 8h.The the expression of apaf1,bax,tnfr1 and fadd showed a certain time and dose effect by incubated with different concentrations of Z-VAD-FMK.In summary,compared with the others two pathways,the mitochondrial apoptotic pathway may play a more important role in the apoptosis of Monopterus albus oocytes.These results provide a basic data for further study on the reversal mechanism in Monopterus albus.