CHARACTERIZATION AND EXPRESSION OF INSULIN-LIKE GROWTH FACTOR 3 (IGF3) GENE IN REGULATING VITELLOGENESIS OF BONY-LIP BARB (OSTEOCHILUS VITTATUS VALENCIENNES, 1842)

• Main Author: Madihah
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66042
• Institution: Institut Teknologi Bandung

Bony-lip barb is an endemic fish of Indonesia, which is used for consumption and as a bio-control agent, so it has the potential to be cultivated. The main concern in the bony-lip barb farming is unsustainable production due to low egg quality. Egg quality depends on various factors that accumulate during vitellogenesis and are important for embryonic development. Vitellogenesis in the process of collecting egg yolk precursor protein, especially vitellogenin (Vtg), by growing oocytes for processing and storage in the ooplasm. The main regulator of vtellogenesis is estradiol-17? (E2), however, Igf is also play a role. Igf3, one of the Igf ligands specifically found in fish, has been reported to play a role in gonadal development, oocyte maturation and ovulation, and steroidogenesis in the ovaries, as well as the regulation of metabolic shift, from growth to reproductive mode. Information regarding the involvement of the igf3 gene in the regulation of vitellogenesis and its relationship in determining egg quality is not available yet. This study aims to (i) characterize the igf3 gene in O. vittatus and determine the homology between Igf3 peptide in Teleost; (ii) observe the igf3 gene expression profile during ovarian development and maturation, as well as the reproductive performance in one reproductive cycle of the female brood; and (iii) determine the relationship between the igf3 gene expression and other genes involved in the regulation of vitellogenesis on the egg quality. The study is divided into 3 stages. In the first study (1), we identified a full-length cDNA of 1081 nucleotides encoding 198 amino acids (aa) of the open reading frame (ORF). In coding sequences (CDS), there is a conserved domain of the IIGF-like superfamily consisting of 68 aa that comprise of domains B, C, A, and D in mature Igf3 peptides as well as six cysteines (C) aa in domains B and A that was a conserved motif in the protein-insulin superfamily. The Igf3 aa deduced from the cDNA sequences showed a high homology (74–92%) with Igf3 proteins from other Cyprinids. Phylogenetic tree construction revealed that the O. vittatus Igf3 is closely related to Igf3b of Common carp (Cyprinus carpio) and forms a monophyletic group with Igf3 from other Cyprinids, while Igf3 from non-Cyprinid species forms a paraphyletic group. In the second study (2) we found that Igf3 mRNA was highly expressed in whole blood and ovaries, moderately expressed in liver, gills, kidneys, heart, and intestines, and the lowest in pituitary and muscle (p<0.05). Relative expression of igf3 mRNA increased significantly throughout the ontogeny gonadal development from embryonic stage to adult with matured ovary (p<0.05). During one reproductive cycle (118 days), the relative expression of igf3 mRNA showed a high and positive correlation (r = 0.903; p<0.01) with the gonadosomatic index, which increased in the developing phase (days 1 to 29) and reach a peak at spawning-capable phase (days 43 to 113) along with the oocyte growth and maturation, then decreased significantly in the regression (day 114) and regeneration phases (day 116 and 118) (p<0.05). Egg quality, which was observed based on the seed performance was influenced by the duration of the spawning-capable phase. The longer the duration of the spawning-capable phase up to 85 or 115 days, then the egg diameter, the fertilization rate, the hatching rate, the larval survival at 3 dph, and the percentage of abnormal larvae were significantly increased (p<0.05). However, the absolute fecundity, the hatching rate, and the larval survival at 10 dph did not significantly differ compared with the duration of 57 days (p>0.05). In the third study (3) E2 treatment at doses of 0 (K), 105 (P1), 210 (P2), or 420 (P3) ?g/kg BW was carried out to observed the regulation of vitellogenesis in O. vittatus. The E2 treatment mainly modified the expression of the igf3 gene and other genes involved in the regulation of vitellogenesis, namely vtgr, esr1, esr2a, and esr2b, depend on dose, time, and reproductive phase. At 24 h, E2 treatment significantly decreased the relative expression of igf3 and vtgr mRNA, otherwise the expression of esr1 and esr2b increased compared to control (p<0.05). At 72 hours of early developing phase of oocytes, the relative expression of igf3 mRNA was positively correlated with vtgr but negatively correlated with esr1 and esr2b. During the next phase of oocyte development, the relative expression of vtgr, esr1, and esr2a mRNA decreased, however, the igf3 mRNA expression was increased until it peaked the spawningcapable phase (p<0.05). The seed performance resulted by the artificial spawning at day 57 depends on the doses of E2 treatment. Absolute fecundity and egg diameter were observed to be the highest in P2 treatment; fertilization percentage, hatching percentage, and survival rate of the 3 dph larvae were the lowest in P3 treatment; and the lowest number of abnormal larvae were observed in the P1 treatment, which was significantly different from the control (p<0.05). In conclusions, the characteristics of igf3 gene from O. vittatus are similar to the Igf3 protein from other Teleost and the closest homology was to Igf3b from C. carpio. Igf3 gene may play an important role in gonadal development and ovarian maturation in the female Bonylip barb. The increase in the relative expression of igf3 mRNA during the ovarian development and maturation stage corresponds to the relative expression profile of vtgr and esr1 mRNA, indicates that the igf3 gene plays a role in the upregulation of vitellogenesis that indirectly affects the egg quality by a suggested mechanism that is involved the regulation of steroidogenesis in the ovaries especially E2 as the main regulator of vitellogenesis, as well as the regulation of metabolic shift from growth to reproductive mode during vitellogenesis.