Identification of Insulin-like Growth Factor (IGF) Family Genes in the Golden Pompano, Trachinotus ovatus: Molecular Cloning, Characterization and Gene Expression

Abstract

Insulin-like growth factors (IGFs) are hormones that primarily stimulate and regulate animal physiological processes. In this study, we cloned and identified the open reading frame (ORF) cDNA sequences of IGF family genes: the insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), and insulin-like growth factor 3 (IGF3). We found that IGF1, IGF2, and IGF3 have a total length of 558, 648, and 585 base pairs (bp), which encoded a predicted protein with 185, 215, and 194 amino acids (aa), respectively. Multiple sequences and phylogenetic tree analysis showed that the mature golden pompano IGFs had been conserved and showed high similarities with other teleosts. The tissue distribution experiment showed that IGF1 and IGF2 mRNA levels were highly expressed in the liver of female and male fish. In contrast, IGF3 was highly expressed in the gonads and livers of male and female fish, suggesting a high influence on fish reproduction. The effect of fasting showed that IGF1 and mRNA expression had no significant difference in the liver but significantly decreased after long-term (7 days) fasting in the muscles and started to recover after refeeding. IGF2 mRNA expression showed no significant difference in the liver but had a significant difference in muscles for short-term (2 days) and long-term fasting, which started to recover after refeeding, suggesting muscles are more susceptible to both short-term and long-term fasting. In vitro incubation of 17β-estradiol (E₂) was observed to decrease the IGF1 and IGF3 mRNA expression level in a dose- (0.1, 1, and 10 μM) and time- (3, 6, and 12 h) dependent manner. In addition, E₂ had no effect on IGF2 mRNA expression levels in a time- and dose-dependent manner. The effect of 17α-methyltestosterone (MT) in vitro incubation was observed to significantly increase the IGF3 mRNA expression level in a time- and dose-dependent manner. MT had no effect on IGF2 mRNA but was observed to decrease the IGF1 mRNA expression in the liver. Taken together, these data indicate that E₂ and MT may either increase or decrease IGF expression in fish; this study provides basic knowledge and understanding of the expression and regulation of IGF family genes in relation to the nutritional status, somatic growth, and reproductive endocrinology of golden pompano for aquaculture development.

Keywords: IGFs; Trachinotus ovatus; cloning; fasting; fish reproduction; sex steroids.

Figure 1

Nucleotide and deduced amino acid sequences of golden pompano IGF1 (A), IGF2 (B), and IGF3 (C). The signal peptide is underlined. The mature region is highlighted with grey color; the nucleotides and amino acids are numbered from right to left. The stope codon is marked by an asterisk (*).

Figure 1

Nucleotide and deduced amino acid sequences of golden pompano IGF1 (A), IGF2 (B), and IGF3 (C). The signal peptide is underlined. The mature region is highlighted with grey color; the nucleotides and amino acids are numbered from right to left. The stope codon is marked by an asterisk (*).

Figure 2

Multiple alignments of amino acid sequences of IGF1 (A), IGF2 (B), and IGF3 (C). Amino acid numbers are indicated on the right side of the sequences, all domains have been boxed and indicated as B, C, A, D, and E, and asterisk (*) indicates position that have fully conserved residues (identical AA residues between sequences), colon (:) indicates variation in AA at some sites (closely related AA), dot (.) indicates more than one variation in AA (distantly related AA). The DNAStar software used to construct multiple sequence alignment. See Table S1 for species name and NCBI GenBank accession numbers.

Figure 3

A phylogenetic tree of IGF1, IGF2, and IGF3 between golden pompano and other teleosts. The values at nodes in the system tree represent the confidence of the bootstrap test of 1000 evaluations. The evolutionary history was inferred using the neighbor-joining method. (“●”) represents Trachinotus ovatus, and bootstrap values (%) are indicated (1000 replicates). Amino acid sequence percentage similarity of the IGF family genes in golden pompano and other teleosts. MEGA11 was used to conduct the evolutionary analysis. See Table S1 for the species name and NCBI GenBank accession numbers.

Figure 4

Tissue expression arrays of IGF1 (A), IGF2 (B), and IGF3 (C) in the golden pompano. Amplification of β-actin was used as the in-house regulator. The statistical significance was calculated using two-way analysis of variance (ANOVA), followed by Tukey’s test (p < 0.05). Data are presented as mean ± standard error (SEM). Asterisk (*) indicates a significant difference (**** p < 0.0001, * p = 0.0069).

Figure 5

Effects of fasting on mRNA expression of IGF1 and IGF2 in the liver and muscle of golden pompano: (A) IGF1 in the liver, (B) IGF1 in muscle, (C) IGF2 in the liver, (D) IGF2 in muscle. Amplification of β-actin was used as the in-house regulator. Data are represented as mean ± SEM (n = 8). A two-way ANOVA and Turkey’s test determined significant differences (p < 0.05). Asterisk (*) indicates significant difference (* p < 0.0278, ** p < 0.0067). (ns) indicates nonsignificant.

Figure 6

Time and dose-dependent in vitro effects of E₂ on mRNA expression of IGF genes in the liver and ovary of golden pompano: (A) IGF1, (B) IGF2, and (C) IGF3. Amplification of β-actin was used as the in-house regulator. Data are presented as mean ± standard error (SEM) (n = 3), and the statistical significance (compared to the control group) was determined using two-way ANOVA, followed by Tukey’s test (p < 0.05). (*) indicates significant differences (* p < 0.01, ** p < 0.0025, *** p < 0.0005). (ns) indicates nonsignificant.

Figure 7

Time and dose-dependent in vitro effects of MT on mRNA expression of IGF genes in the liver and ovary of golden pompano: (A) IGF1, (B) IGF2, and (C) IGF3. Amplification of β-actin was used as the in-house regulator. Data are presented as mean ± standard error (SEM) (n = 3), and the statistical significance (compared to the control group) was determined using two-way ANOVA, followed by Tukey’s test (p < 0.05). (*) indicates significant differences (** p < 0.0021, **** p < 0.0001). (ns) indicates nonsignificant.