Growth and differentiation factor (GDF9) in the ovary of zebrafish, Danio rerio

The intercommunication between the somatic follicle cells (granulosa and theca cells) and oocyte is essential for normal growth and development of ovarian follicle, and oocytes play central roles in promoting follicle growth. The oocyte achieves its function partly by secreting paracrine growth factors that act on their surrounding follicle cells. Growth and differentiation factor 9 (GDF9) is a member of TGFbeta superfamily. It was first identified as an oocyte-specific growth factor in mammals. GDF9 is critical for follicle development as demonstrated by GDF9 knock-out mouse or mutant sheep, whose follicle developments were blocked at primary stage. In mammals, GDF9 is involved in different physiological processes including folliculogenesis, steroidogenesis and cumulus expansion in the ovary. However, the information about GDF9 in non-mammalian animals is rather limited. Using zebrafish as a model, I have undertaken the present study to address a series of issues about GDF9.;The zebrafish gdf9 was first cloned, and amino acid sequence analysis showed that the mature region of Gdf9 shared high homology with their counterparts in mammals with six cysteines full conserved. In zebrafish, gdf9 mRNA was only detected in the ovary and testis by RT-PCR analysis. Northern blot analysis demonstrated only one transcript in the ovary. The expression of gdf9 mRNA was further localized in the oocyte. The oocyte-specific feature is consistent with that in most mammals.;In zebrafish ovarian follicles, the expression of gdf9 gradually decreased with the development of follicles from primary growth to full-grown immature stage as demonstrated by real-time RT-PCR. Consistently, in situ hybridization showed that the PG follicles had the strongest staining for gdf9 mRNA. Zebrafish gdf9 maintained its expression after fertilization until early embryonic stage. Its expression significantly decreased when embryo entered the gastrula period and disappeared at shield stage.;Zebrafish is a juvenile hermaphrodite and adult gonochorist, and its gonad develops as ovary-type first, followed by either developing as the ovary or differentiating into the testis after oocyte degeneration. The earliest time to detect expression of gdf9 again was 17 day post-fertilization (dpf) by RT-PCR. Interestingly, the expression of gdf9 was highly correlated with gonadal sex differentiation with distinct sexually dimorphic patterns in larval and juvenile stage. The expression of gdf9 was strong if the gonad developed as ovary, but its expression was undetectable or very low in those altered ovary or newly formed testis.;The regulation of gdf9 by extracellular signals such as gonadotropins and ovarian growth factor was also studied. Its expression was down-regulated by hCG both in vivo and in vitro . When tested on immature full-grown follicles, the inhibitory effect of hCG on gdf9 expression was both time and dose-dependent. Consistently, db-cAMP and forskolin, which increase cAMP level, decreased gdf9 expression in the immature full-grown follicles. Furthermore, the inhibitory effect of hCG also seemed to be stage-dependent. The expression of gdf9 in early vitellogenic and pre-vitellogenic follicles did not seem to be affected by hCG;Besides gonadotropin, activin, an ovarian growth factor was also shown to suppress gdf9 expression in vitro. In zebrafish, oocyte is likely the target of activin action and gdf9 may be one of the downstream targets of activin in the oocyte. After applying the conditioned Gdf9 medium from recombinant mammalian cells, the expression of activin/inhibin subunit betaA (inhba) and betaB (inhbb) seemed to be up-regulated. There may exist a regulatory loop between Gdf9 from oocyte and activin from the follicle layer.;The present study has provided clear evidence that Gdf9 is a secreted oocyte-specific growth factor in the zebrafish, and its function is closely related to follicle development as well as sex differentiation in the zebrafish.