Abstract
During the early stage of folliculogenesis, known in fish as previtellogenesis, the oocyte develops competence for the accumulation of yolk proteins in the ooplasm later in development, during the vitellogenic stage. The yolk proteins are essential for embryonic and early larval development post-fertilization. Precise coordination between the oocyte and adjacent somatic cells (i.e., granulosa and theca cells) is required for development of this competence during the previtellogenic stage. Among the numerous key signaling molecules produced by the oocyte, growth differentiation factor-9 (GDF9) is amongst the best-understood factors involved in regulating ovarian folliculogenesis in mammals and chicken; in these animals, its primary function is to stimulate granulosa cell proliferation and differentiation. However, there are striking differences in the reproductive phenotypes of these species. A previous study in our laboratory has identified and localized Gdf9 in the shortfinned eel ovary, but its target tissue and function remain to be elucidated. The present study, therefore, aimed to identify and localize the receptors that mediate the action of Gdf9, i.e., activin receptor-like kinase-5 (Alk5) and bone morphogenetic protein receptor type II (Bmpr2), and shed light on the role of Gdf9 and its regulation during early folliculogenesis in the shortfinned eel ovary.
Firstly, alk5 and bmpr2 genes were isolated, characterized and their transcript abundances during early folliculogenesis quantified by quantitative real-time PCR (Chapter 2). As expected, transcript abundance of both receptors in the ovary increased from the previtellogenic to early vitellogenic stage along with that of their ligand. Secondly, specific antibodies for Alk5 and Bmpr2 were produced and subsequent analysis revealed that Alk5 and Bmpr2 are located in the somatic cells of shortfinned eel follicles. Localization of transcripts of these receptors was reinforced by in situ hybridization (Chapter 3). These findings, together with the ovarian expression pattern of Gdf9, strongly suggest paracrine signaling of Gdf9 in the shortfinned eel ovary during early folliculogenesis. Thirdly, to examine whether Gdf9 is essential for follicular development in fish, the role of Gdf9 in the shortfinned eel ovary was investigated using a knockdown approach through immunoneutralization of Gdf9. The immunoneutralization experiment failed to expose the role of Gdf9 in shortfinned eel ovary and thus, Gdf9 function in fish remains unknown (Chapter 4). Lastly, the effects of several hormones (i.e., gonadotropins, growth factors, steroids or metabolic hormones) that reflect reproductive/metabolic status on the expression of Gdf9 and its receptors in the shortfinned eel ovary during early folliculogenesis were examined (Chapter 5). To do this, the shortfinned eel gdf9 promoter was isolated, ligated into a luciferase reporter vector and transfected into human COV434 granulosa cells, followed by treatment with the aforementioned hormones. Furthermore, in vitro effects of a subset of these selected hormones on ovarian gdf9, alk5 and bmpr2 transcript abundance were examined. Quantitative analysis of shortfinned eel gdf9 promoter activity together with analysis of transcript abundance of gdf9, alk5 and bmpr2 showed an effect of all hormones on Gdf9 and/or its receptors in the shortfinned eel ovary. Moreover, incubation of ovarian explants showed that effects of these ligands on gene expression are more profound in early vitellogenic than in late previtellogenic ovarian tissue. Findings of the present study provided valuable insights into the genomic structure of gdf9 and added further evidence to a previous study in shortfinned eel by identification of the target cells of Gdf9 and the regulation of Gdf9 and its receptors during early folliculogenesis.