Foxl2 Up-Regulates Aromatase Gene Transcription in a Female-Specific Manner by Binding to the Promoter as Well as Interacting with Ad4 Binding Protein/Steroidogenic Factor 1

Abstract

Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17beta-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17beta-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.

Gonadal soma-derived factor (gsdf), a TGF-beta superfamily gene, induces testis differentiation in the teleost fish Oreochromis niloticus

“…To evaluate the specificity of the antiserum, pCAGGS containing the ORF of #9475 (see Section 195 2.8) was transfected into human embryonic kidney (HEK) 293 cells according to a method described elsewhere (Zhou et al, 2007). Incubation media were collected at 1 and 2 days after the transfection and subjected to immunoblot analysis.…”

“…In vivo transgenic overexpression of the #9475 gene in XX individuals was performed according to the methods of our previous study (Wang et al, 2007). An expression plasmid for #9475 was produced as follows.…”

“…Conversely, the downstream cascade of genes involved in gonadal sex differentiation has been thought to be relatively conserved among diverse species (Capel, 2000), although differences in downstream regulatory networks between mammals and medaka were 35 recently pointed out (Herpin et al, 2013). Downstream genes such as dmrt1 (doublesex/mab-3-related transcription factor-1) (Adolfi et at., 2015, Guan et al, 2000Shibata et al, 2002;Smith et al, 1999;Raymond et al, 1998Raymond et al, , 1999a, sox9 (Capel, 2000;Kobayashi et al, 2008;Nakamoto et al, 2005;Vidal et al, 2001), amh (anti-Mullerian hormone) (Cutting et al, 2013), wt1 (Chen et al, 2013;Takasawa et al, 2014), and foxl2 (Baron et al, 2004;Pailhoux 40 et al, 2002Pailhoux 40 et al, , 2001Ottolenghi et al, 2005;Wang et al 2007) have been investigated in many vertebrate species, but the overall gene network and its regulation remain unclear.…”

“…A major locus on the Y chromosome has been shown to control sex determination, but no sex-determining gene has yet been identified in tilapia (Cnaani et al, 2008;Volff et al, 2007). However, the expression patterns of most of the downstream genes involved 55 in gonadal sex differentiation, especially those of foxl2, cyp19a1a (P450arom), fshr, hsd17b1, dmrt1, and sox9a, have been investigated (Ijiri et al, 2008;Kobayashi et al, 2008Kobayashi et al, , 2003Wang et al 2007;Yan et al, 2012). In light of these studies, the period before/at 5-6 days post-hatching (dph) is critical to the initiation of sex determination/differentiation, in which undifferentiated gonads differentiate into testes or ovaries in tilapia.…”

Characterization of early molecular sex differentiation in rainbow trout, Oncorhynchus mykiss

“…In the mouse, the inactivation of Foxl2 (Ottolenghi et al, 2005), Fst (Jorgez et al, 2004), or Cyp19 (Britt et al, 2002) leads to the development of Sertoli cell tubule-like structures in female gonads. In addition, recent work in the goat (Pannetier et al, 2006) and in the tilapia (Wang et al, 2007) also suggests that Foxl2 may be a direct transcriptional activator of Cyp19. Considering this and the tight positive temporal correlation observed between the expression profiles of cyp19a1, fst, foxl2a, and inha during early ovarian differentiation in trout, a close interaction of these genes for the maintenance of granulosa cell differentiation and/or estrogen production may be hypothesized.…”