Proteomic analysis of zebrafish folliculogenesis identifies YB-1 (Ybx1/ybx1) as a potential gatekeeping molecule controlling early ovarian folliculogenesis.

As in mammals, ovarian folliculogenesis in teleosts also consists of two phases: the primary growth (PG) and secondary growth (SG) phases, which are analogous to the preantral and antral phases respectively in mammals. In this study, we performed a proteomic analysis on zebrafish follicles undergoing the PG-SG transition aiming to identify factors involved in the event. Numerous proteins showed significant changes, and the most prominent one was Y-box binding protein 1 (YB-1; Ybx1/ybx1), a transcription factor and mRNA-binding protein. YB-1 belongs to the Y-box binding protein family, which also includes the gonad-specific YB-2. Interestingly, phylogenetic analysis showed no YB-2 homolog in zebrafish. Although ybx1 mRNA was expressed in various tissues, its protein Ybx1 was primarily produced in the gonads, similar to YB-2 in other species. In the ovary, Ybx1 protein started to appear in early follicles newly emerged from the germ cell cysts, reached the highest level in late PG oocytes, but decreased precipitously when the follicles entered the SG phase. In PG follicles, Ybx1 might function as a key component of the messenger ribonucleoprotein particles (mRNPs) in association with other RNA-binding proteins. Similar to mammalian YB-1, zebrafish Ybx1 also contains functional signals that determine its intracellular localization. In conclusion, Ybx1 may play dual roles of YB-1 and YB-2 in zebrafish. In the ovary, Ybx1 binds mRNAs to stabilize them while preventing their translation. At PG-SG transition, Ybx1 is removed to release the masked mRNAs for translation into functional proteins, leading to follicle activation.

Separation of Oocyte and Follicle Layer for Gene Expression Analysis in Zebrafish.

Zebrafish ovarian follicles are mainly composed of the oocyte and a thin layer of follicle cells. Recent studies have demonstrated extensive cell-cell interactions between the oocyte and surrounding follicle layer and that the two compartments communicate mostly through paracrine factors. To understand the paracrine communication within the follicle, it is essential to know the spatial expression patterns of genes in the two compartments. However, since the follicle layer is extremely thin and the oocytes are enormous in size in fish, it is often difficult to detect gene expression by traditional methods such as in situ hybridization. Separation of the oocyte and surrounding follicle layer followed by RT-PCR detection provides a sensitive way to reveal the expression of individual genes in the two compartments of the follicle. This chapter introduces a method for mechanic separation of the oocyte and follicle layer at full-grown stage for expression analysis. Since fish have similar follicle structure, this method may also be used in other species as well.

Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation.

Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.