Follistatin, an Early Player in Ovarian Differentiation in Teleost Fish?

Abstract

Follistatin (Fst) is an inhibitor of some members of the TGFβ family that was first identified in mammalian ovary. Fst gene has been shown to be expressed in granulosa cells and oocyte of mammalian and fish species, and is involved in folliculogenesis and oocyte development. In addition to these functions in ovarian development, Fst is already expressed in the mouse embryonic ovary, and its inactivation induces partial female-to-male sex reversal, suggesting that Fst plays a significant role in early ovarian differentiation in mammals. However, only little is known about its potential role in early gonadal differentiation in teleost fish. Rainbow trout (Oncorhynchus mykiss) is a teleost fish with a strict male heterogametic sex determination system (XX/XY). Genetic all-male (XY) and all-female (XX) experimental populations can be produced in that species, allowing the sex-specific characterization of the early molecular events that occur before histological differentiation of the gonads. We then used the rainbow trout model to investigate the expression pattern of fst in embryonic gonads by WHISH, and to determine its regulation during sex differentiation in fish. Shortly after hatching that occurs around 30 days post-fertilization (30 dpf), fst is already expressed in female gonads in some clusters of somatic cells, whereas no expression is detected in male gonads. Later during ovarian differentiation, its expression is restricted in some somatic cells lining the future ovarian lamellae. This expression profile is very similar to what is observed for the aromatase gene (cyp19a1a). Given that cyp19a1a is coding for a key steroidogenic enzyme needed for ovarian differentiation in fish and also the earliest known gene to be expressed in a sexually dimorphic fashion in female gonads of rainbow trout, we compared fst and cyp19a1a expressions several days before hatching. Both fst and cyp19a1a expression begins around 26 dpf in somatic cells, in a very particular punctiform pattern throughout the female gonads. Then, around hatching, this expression becomes continuous throughout the gonads. These results suggest that fst is implicated along with cyp19a1a in the early steps of ovarian differentiation in teleost fish. As fst expression is known to be induced by the Wnt/β-Catenin pathway in mammals, we treated embryos in vivo with lithium chloride (LiCl), an activator of Wnt pathway, and we analysed by qPCR the effects of this treatment on fst expression. We found that these LiCl treatments increase fst expression, suggesting a role of the Wnt/β-Catenin pathway in regulating expression of fst in rainbow trout.These results demonstrate that follistatin is one of the earliest markers of ovarian differentiation in rainbow trout and its co-expression with cyp19a1a suggests that fst could be implicated in the regulation of estrogen production. Furthermore, expression of fst is upregulated by the Wnt/β-Catenin pathway, suggesting a wide conservation of fst regulation between mammals and fish. (platform)