PARENT SESSION

(W486) CLONING AND CHARACTERIZATION OF ZEBRAFISH GROWTH AND DIFFERENTIATION FACTOR-9 (GDF-9) AND ITS REGULATION BY GONADOTROPIN IN THE OVARY.

Liu, Lin¹, Ge, Wei¹, ¹ The Chinese University of Hong Kong, Shatin, Hong Kong

ABSTRACT- Growth and differentiation factor-9 (GDF-9) is a member of transforming growth factor (TGF-) superfamily. As an oocyte-specific growth factor, GDF-9 plays critical roles in controlling folliculogenesis in mammals. But so far, its existence and function in non-mammalian vertebrates remain largely unknown. In the present study, we cloned a cDNA (2100 bp) of zebrafish GDF-9 homolog (gdf9), which shows about 60% homology with that of mammals in the mature region. RT-PCR analysis showed that GDF-9 expression could only be detected in the gonads, and Northern blot analysis revealed one single transcript (about 2.0 kb) in the ovary. Real-time RT-PCR analysis demonstrated that GDF-9 had the highest expression in the primary growth follicles, and its expression level gradually decreased with follicle development with the lowest level detected in the full-grown follicles. The expression of GDF-9 maintained through fertilization and early embryonic development until gastrulation when the expression level dramatically decreased. The expression became undetectable after the pharyngula stage. Within the follicle, GDF-9 was localized predominantly in the oocytes as demonstrated by RT-PCR on denuded oocytes and isolated follicle layers as well as in situ hybridization. Interestingly, the follicle layers also expressed GDF-9 albeit at much lower levels. This is further supported by the evidence that the expression could be detected in cultured zebrafish follicle cells that were free of oocytes. The expression of GDF-9 could be down-regulated by hCG and goldfish pituitary extract in both ovarian fragments and isolated follicles in a dose and time-dependent manner, and the inhibition appeared to be stage-dependent with the strongest inhibition observed in the full-grown follicles and no effect in the primary growth follicles. This correlates well with the expression profiles of LH receptor in the zebrafish follicles. Interestingly, the expression of GDF-9 in the cultured follicle cells also responded to hCG, but in an opposite manner with hCG enhancing its expression. In conclusion, as an oocyte-derived growth factor, GDF-9 is well conserved across vertebrates. With its biological advantages, zebrafish will surely provide an outstanding model for further studying its function and regulation.

KEY WORDS: GDF-9, oocyte, zebrafish