ELIMINATION OF GERM CELLS ON GONADAL DIFFERENTIATION IN PELODICUS SINENSIS

In reptiles, primordial germ cells proliferate massively in the female cortex and entry the “mitotic arrest” in the male medulla when they reach the genital ridge. Somatic cells facilitate germ cells development and differentiation. However, it is not clear how germ cells affect somatic cells and whether germ cells depletion could influence the sex differentiation. In order to investigate whether germ cells have impacts on somatic cells and gonadal sex differentiation, we depleted the endogenous germ cells in ZZ and ZW genetic Pelodicus sinensis by injecting of 20 mg/kg busulfan into the embryos in this study. Immunofluorescence of VASA showed that the germ cells are depleted after treatment of busulfan in both ZZ and ZW gonads. H & E staining analysis showed that the morphology of cortex and medullary cords had no difference with or without busulfan treatment in ZZ and ZW turtles. The busulfan treated ZW gonads expressed the ovary-specific marker FOXL2 and CYP19A1, while the busulfan treated ZZ gonads showed immunofluorescence staining of testicular marker protein DMRT1 and SOX9. The Real-time PCR analysis showed that the busulfan-treated ZW gonads still expressed high levels of Cyp19a1 and Foxl2 and low levels of Dmrt1 and Sox9, while the busulfan-treated ZZ gonads expressed high levels of Dmrt1 and Sox9 and relative low levels of Cyp19a1 and Foxl2. These results suggest that the gonads sex do not change after the depletion of germ cells in both ZW and ZZ genetic turtles. In conclusion, our study first proved that the germ cells depletion do not alter the sex differentiation in GSD reptiles, which can provide reference significance for breeding of Pelodicus sinensis.