Molecular Cloning, Gene Expression Of Foxl2 And Its Possible Roles In Gonadal Sex Differentiation In Southern Catfish, Silurus Meridionnalis Chen

Foxl2 (winged helix/forkhead transcription factor gene 2), a member of the forkhead (FH) gene family, is a newly isolated transcription factor expressed specifically in the eyelid and adult ovary. In human and mammals, Foxl2 plays a crucial role in female reproduction, especially in differentiation and proliferation of granulosa cells, ovarian development and maintenance of ovarian function by regulating the transcription of target genes.To address the role of Foxl2 in sex determination and differentation in Southcatfish, the cDNA(GenBank Accession No. EF015396) was isolated from the gonad by reverse transcription polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). The Southern catfish Foxl2 cDNA contains 1455 bp encompassing a 5'UTR of 228bp, a coding region of 900bp and a 3'UTR of 327 bp including the poly (A) tail. The open reading frame encodes a protein of 299 amino acid, which contains a typical DNA binding domain (FH-Domain) shared by all members of the forkhead family. Phylogenetic analysis of known Foxl2 sequences from various vertebrates revealed that Southern catfish Foxl2 is highly conserved in the FH-Domain. Outside the FH-domain, the C-terminal region is more conserved than the N-terminal region. However, like other non-mammalian Foxl2s, the Southern catfish Foxl2 contains neither the 14-polyalanine tract nor the glycine and proline repeats, which are present in all mammalian Foxl2 sequences.Semi-quantitative RT-PCR analysis was performed to examin the expression pattern of Fox/2 in the male and female catfish. The results showed that Foxl2 was expressed exclusively in the brain (B), pituitary (P), gill and gonads (G), with the highest level in the ovary, reflecting the possible involvement of Faxl2 in the B-P-G axis.All-female Southern catfish fry were treated with aromatase inhibitor fadrozole (F), estrogen receptor antagonist tamoxifen (TAM) and estrogen 17β-estradiol (E₂), respectively, from 5 to 25 days after hatching (dah). The expression levels of Foxl2, ovarian type aromatase Cyp19a and brain type aromatase Cyp19b were measured at 65dah. In the F, TAM and F+TAM treated groups, Foxl2 and Cyp19a were down-regulated in the gonad, while the expression of Cyp19b in the gonad remained unchanged. Furthermore, down-regulation of Foxl2 and Cyp19b was also detected in the brain. In the E₂ treated group, Foxl2 and Cyp19b were up-regulated in the brain. Consistent with the observed changes in the expressions of these genes, 56%, 70% and 80% sex reversed male individuals were obtained in the F, TAM and F+TAM treated groups, respectively. At 120dah, gonads of the drug treated fish were analyzed histologically. Partial and complete sex reversal was observed in the F, TAM and F+TAM treated groups with different appearance and features. The ovarian walls of these fish were thickened and the ovarian cavity shrinked. In the F treated group, many degenerating follicles were observed, and the oocyte number was decreased because of degeneration of the follicles, while the numbers of somatic cells were increased as compared with the control. In the TAM treated group, some cavums which might be the space left behind by the degenerated follicles, were also observed. Moreover, elongated ellipse-shaped oocytes which are different from the round- or ellipse-shaped oocytes in the control group were also observed. In the F+TAM treated group, spermatocyst-like structure, spermatogonia and primary spermatocytes were observed. On the other hand, the gonad histology of the E₂ treated fish was the same as the control group. These results show that Foxl2, Cyp19a and Cyp19b are involved in the sex differentation in Southern catfish at the brain and/or gonad levels.