Molecular cloning and expression analysis of fushi tarazu factor 1 in the brain of air-breathing catfish, Clarias gariepinus

Abstract

Background: Fushi tarazu factor 1 (FTZ-F1) encodes an orphan nuclear receptor belonging to the nuclear receptor family 5A (NR5A) which includes adrenal 4-binding protein or steroidogenic factor-1 (Ad4BP/SF-1) and liver receptor homologue 1 (LRH-1) and plays a pivotal role in the regulation of aromatases.

Methodology/principal findings: Present study was aimed to understand the importance of FTZ-F1 in relation to brain aromatase (cyp19a1b) during development, recrudescence and after human chorionic gonadotropin (hCG) induction. Initially, we cloned FTZ-F1 from the brain of air-breathing catfish, Clarias gariepinus through degenerate primer RT-PCR and RACE. Its sequence analysis revealed high homology with other NR5A1 group members Ad4BP/SF-1 and LRH-1, and also analogous to the spatial expression pattern of the latter. In order to draw functional correlation of cyp19a1b and FTZ-F1, we analyzed the expression pattern of the latter in brain during gonadal ontogeny, which revealed early expression during gonadal differentiation. The tissue distribution both at transcript and protein levels revealed its prominent expression in brain along with liver, kidney and testis. The expression pattern of brain FTZ-F1 during reproductive cycle and after hCG induction, in vivo was analogous to that of cyp19a1b shown in our earlier study indicating its involvement in recrudescence.

Conclusions/significance: Based on our previous results on cyp19a1b and the present data, it is plausible to implicate potential roles for brain FTZ-F1 in ovarian differentiation and recrudescence process probably through regulation of cyp19a1b in teleosts. Nevertheless, these interactions would require primary coordinated response from ovarian aromatase and its related transcription factors.

Figure 1. ClustalW alignment of deduced amino acid sequence of catfish FTZ-F1 with other vertebrate counter parts.

The alignment was done using software ClustalW (EBI tools). Shaded region represents conserved amino acids and signature domains are represented by rectangle boxes. GenBank accession numbers of the sequences we used are as follows: Clarias gariepinus; JN859075, Ictalurus punctatus; DQ000612, Oreochromis niloticus; AB060814, Oryzias latipes; AB016834, Oncorhynchus mykiss; NM_001124537, Danio rerio; NM_131463, Acanthopagrus schlegeli; AY491379, Taeniopygia guttata; NM_001076692, Pelteobagrus fulvidraco; EU860284, Xenopus laevis; BC169770, Gallus gallus; NM_205077, Mus musculus; AF511594 and Homo sapiens; BC118571.

Figure 2. Phylogenetic tree showing the evolutionary status of catfish FTZ-F1.

Phylogenetic tree constructed using NJ method and bootstrap analysis with 1000 replicates was used to assess the strength of nodes in the tree. Phylogenetic analysis was done using ClustalW software of DNA data bank of Japan and supported by Tree view software. The scale bar represents 0.1 substitutions per amino acid site. GenBank accession numbers of the sequences used in the analysis are indicated in Fig. 1.

Figure 3. Peptide affinity purification profile of FTZ-F1 antibody and Western blot analysis.

A) IgG fraction of FTZ-F1 antibody showing band at ∼55 kDa (heavy chain) and ∼25 kDa (light chain). B) No signal was seen in negative control. C) A protein band at ∼45 kDa corresponding to deduced FTZ-F1 protein detected in the female brain protein homogenate of preparatory phase catfish.

Figure 4. Tissue distribution of FTZ-F1 in adult female catfish.

A) qRT-PCR analysis of FTZ-F1 expression (reported as 2^−ΔCT) was done in different tissues of prespawning phase female catfish along with testis of prespawning phase male catfish. Values are mean ± SEM, n = 5. Means with different letters differ significantly and are compared group-wise (P<0.05). B) Tissue distribution pattern of FTZ-F1 by Western blot. As explained above, different tissues of female catfish and testis with FTZ-F1 antibody in upper lane, α-tubulin antibody in lower lane as control to show equal loading.

Figure 5. Catfish brain FTZ-F1 expression during gonadal ontogeny by qRT-PCR.

Quantitative analysis of brain FTZ-F1 expression relative to β-actin expression during gonadal ontogeny of male and female catfish is reported as 2^−ΔCT. Values are mean ± SEM, n = 5. *indicates the significance at P<0.05, ^adenotes the significance at P<0.05 from 50/75 - 300 dph to adult for respective sex.

Figure 6. Expression of FTZ-F1 in brain during different phases of catfish ovarian cycle.

Quantitative analysis of brain FTZ-F1 expression relative to β-actin expression during different phases of ovarian cycle was reported as fold change relative to preparatory phase calculated using 2^−ΔΔCT method. Values are mean ± SEM, n = 5. Means with different letters differ significantly and are compared group-wise (P<0.05).

Figure 7. Expression of FTZ-F1 in catfish brain at different time points after hCG induction, in vivo.

Quantitative analysis of brain FTZ-F1 expression relative to β-actin expression in A) Preparatory and B) Prespawning phases of ovarian cycle compared to saline treated controls by qRT-PCR was reported as fold change relative to 0 h calculated using 2^−ΔΔCT. X-axis represents hours after treatment. Values are mean ± SEM, n = 5. * indicates significance at P<0.05.