Expression of tilapia CREBs during: (A) natural ovarian cycle and (B) hCG induced -oocyte maturation as determined by RT-PCR and Northern Blot respectively. A plasmid DNA with CREB partial cDNA was used as positive control (PC) and PCR reaction without RT was used as negative control (NC) (Ma, Marker). (C) Densitometric analysis of CREB2 and CREB3 (mean ±SEM of two independent experiments).

<p>Expression of tilapia CREBs during: (A) natural ovarian cycle and (B) hCG induced -oocyte maturation as determined by RT-PCR and Northern Blot respectively. A plasmid DNA with CREB partial cDNA was used as positive control (PC) and PCR reaction without RT was used as negative control (NC) (Ma, Marker). (C) Densitometric analysis of CREB2 and CREB3 (mean ±SEM of two independent experiments).</p

Northern blot analysis of CREBs.

<p><b>(A)</b> Expression of CREBs in tilapia ovary and testis <b>(</b>VOF; Vitellogenic Ovarian Follicle; FIO, Full grown Immature Ovarian Follicle; MOF, Mature Ovarian follicle). <b>(B)</b> Representative densitometric analysis of CREB2 and CREB3 of tilapia. Bars depicting same letter are significantly different from each other (mean ±SEM of two independent experiments). <b>(C)</b> Northern blot analysis of CREBs catfish ovary <b>(</b>P, Preparatory; PS, Pre-spawning; S, Spawning). <i>Note</i>: only one form of CREB (~ 1.3 kb) in tilapia testis [CREB 1] while two forms of CREB (~2.85 [CREB 2] and ~2.75 kb [CREB 3]) in tilapia ovary.</p

Clustal W multiple alignment of tilapia and catfish CREB deduced amino acid sequences.

<p>Clustal W multiple alignment of tilapia and catfish CREB deduced amino acid sequences.</p

Tissue distribution pattern of tilapia CREBs as determined by semi-quantitative RT-PCR.

<p>A plasmid DNA of CREB partial cDNA was used as positive control (PC) and PCR reaction without RT was used as negative control (NC). (Ma, Marker; B, Brain; A, Adrenal; H, Heart; S, Spleen; L, Lung; I, Intestine; K, Kidney; M, Muscle; T, Testis; O, Ovary; F, Ovarian follicle).</p

Cladogram showing phylogenetic analysis of the Nile tilapia and catfish CREBs with other vertebrate analogs.

<p>(Accession no.: Human A <u>NM_004379</u>; Human B <u>NM_134442</u>; Mouse C <u>NM_001037726</u>; <i>Xenopus</i><u>NM_001086603</u>;Mouse A <u>NM_009952</u>; Mouse B <u>NM_133828</u>; <u>Rat A NM_ 134443</u>; Rat B <u>NM_031017</u>; Chicken <u>NM_ 204450</u>; Zebrafish <u>NM_ 200909</u>; Pig <u>NM_001099929</u>; Cattle <u>NM_174285</u>; Songbird <u>NM_001048256</u>).</p

Comparison of serum 11-KT levels between <i>Figla</i>-transgene and control XY fish.

<p>Results were presented as means ± S.E.M. of 6 independent samples, respectively.</p

Expression profile of <i>Figla</i> gene in tilapia.

<p>(A) Expression of <i>Figla</i> in adult tilapia tissues by real-time PCR. HK, head kidney; M, muscle; K, kidney; O, ovary; B, brain; T, testis; G, gill; H, heart; L, liver; I, intestine; S, spleen. (B) Ontogenic expression of <i>Figla</i> gene in tilapia gonads analyzed by real-time PCR. Data were represented as means ± S.E.M. of three independent samples. The expression level was normalized using the geometric mean of the levels of three internal control genes (<i>gapdh</i>, <i>ef1a</i>, and <i>actb</i>). Different lower-case letters indicate a significant difference in <i>Figla</i> mRNA levels (P<0.05).</p

Phylogenetic and synteny analysis of <i>Figla</i> gene in vertebrates.

<p>(A) Phylogenetic tree representing the evolutionary relationship among vertebrate homologues of <i>Figla</i>, with tilapia Max (Myc-associated factor X) as an out-group. Values on the tree represent bootstrap scores from 1000 trials, indicating the credibility of each branch. Branch lengths are proportional to the number of amino acid changes on the branch. (B) Synteny maps showing the <i>Figla</i> locus organization in vertebrates. Gene symbols are described according to Ensembl database. The bar lengths are not proportional to the distances between genes. The direction of the arrow indicates the gene orientation.</p

Effects of <i>Fig1a</i> over-expression on subcellular re-organization of somatic and germ cell markers.

<p>No significant difference of Dmrt1, Cyp17a1 and StAR were detected between <i>Fig1a</i>-transgene (A, C, E) and control group (B, D, F). Abundant expression of Sycp3 was detected in the testis of the control fish (H), while no positive immunostaining for Sycp3 was detected in the testis for the <i>Figla</i>-transgene fish (G). Arrows indicate positive immunostaining for each gene.</p

Comparative transcriptional profiling of <i>Figla</i>-transgene and control XY fish.

<p>Expression of <i>dmrt1</i>, <i>prm</i>, <i>cyp17a1</i>, <i>hsd3b1</i> and <i>star</i> in <i>Figla-</i>transgene and control fish at 90 dah were measured by real-time PCR. Relative mRNA levels were represented as means ± S.E.M. of 3 independent samples for the control fish and 5 samples for the <i>Figla</i>-transgene fish. *, indicates the significant difference (P<0.05) between the control and <i>Figla</i>-transgene fish.</p