| The IGF system plays very important roles in diverse physiological processes such as growth, proliferation, survival, migration, and differentiation. Recently, a new member of insulin like growth factor family, named as IGF-3, has been reported from our group. Studies on IGF-3 in tilapia(Oreochromis niloticus) and zebrafish(Danio rerio) indicated the possible involvement of this novel IGF-3 in gonad differentiation, development and maturation. To further understand its role in the Nile tilapia gonad, IGF-3 recombinant protein and its antibody were produced, and three IGF receptors were investigated in this study.Sequence analysis indicates that IGF-3 has a signal peptide (SP) and five domains (B, C, A, D, E), which is similar to IGF-1 and IGF-2. It is known that the mature IGF-1 and IGF-2 peptides consist of B, C, A and D domains, however, it is unclear with IGF-3. Four different expression types of IGF-3 with or without the signal peptide (SP) and E domain were constructed using Pcold I and pGex-4T-1 as expression vectors, respectively. Two types of IGF-3 recombinant protein, named His-IGF-3 (without SP, molecular weight is 21.0 KD) and GST-IGF-3 recombinant protein (without SP and E domain, molecular weight is 33.9 KD) were successfully obtained. His-IGF-3 recombinant protein was purified using Ni-NTA superflow cartridge and used to immunize mouse (Bal b/c. female) three times at 15 days interval with 25-30μg antigen each time. Ten days after the last immunization, mouse blood was collected for ELISA evaluation. The result indicated the IGF-3 polyclonal antibody was functional and had a titre of 1:10000. Both His-IGF-3 and GST-IGF-3 recombinant protein were detected by Western blot using antibodies against His-Tag. GST-Tag and IGF-3 as first antibody. with a dilution factor of 1:1000 and 1:1000 and 1:800, respectively. A band with molecular weight of about 13KD was also detected both in ovary and testis using extracted protein and the obtaind IGF-3 antibody. The result suggested that the mature peptide of IGF-3 consists of B, C, A, D and partial E domain. The expression of IGF-3 protein was also analyzed in tilapia gonad by immunohistochemistry. IGF-3 signal was found in the somatic cells of both ovary (Granulosa cell) and testis (Interstitial cell and Sertoli cell). The purified His-IGF-3 and the IGF-3 antibody were also applied for primary culture of the Nile tilapia ovary segments and were proved to be functionally active.In this study, Three IGF receptors, IGF-IRa, IGF-1Rb (two alternatively splicing isoforms, IGF-1Rb-1S1 and partial IGF-1R-IS2) and IGF-2R cDNAs, were cloned from the Nile tilapia by RT-PCR and RACE. Sequences analysis indicate that both IGF-1Ra and-1Rb possess the tyrosin protein kinase domain, which is critical to the activationof IGF signal pathway, while IGF-2R lack the specified domain. Phylogenetic and functional domain analyses suggested that IGF-1R might share the same ancestor with the insulin receptors, while IGF-2R might have evolved earlier than IGF-1R from another ancestor. RT-PCR was performed to study the tissue distribution of IGF-1Ra, IGF-1Rb and IGF-2R. The highest expression level of IGF-1Ra was found in heart, IGF-1Rb in pituitary, testis and kidney, while the IGF-2R in kidney. Both IGF-1Ra and IGF-1Rb expressed in gonad (ovary and testis) and had a higher expression level in testis than that in ovary, in contrast, no IGF-2R was detected in either ovary or testis. Ontogenic expressions of IGF-1Ra, IGF-1Rb. IGF-3 and IGF-2R in gonads were also analyzed by Real-time PCR. The highest expression level of IGF1Ra in ovary was at 30 dah, then descended till 240 dah, and became lower than the testis from 40 dah. The highest expression level of IGF1Ra in testis was at 50 dah. In contrast, the highest expression level of IGF1Rb in ovary was at 50 dah, then descended till 240 dah and became lower than the testis from 60 dah. The highest expression level of IGF1Rb in testis was at 120 dah. The expression level of IGF-2R was much lower than that of the other two genes, only showing a low expression at early period of 10-70 dah, with the highest expression level in the ovary at 70 dah. became undetectable from 70 dah onwards. These results are consistent with the tissue distribution result of IGF-2R. Like IGF-IRa, IGF-3 also showed the highest expression level in ovary at 30 dah. then descended and became lower than tthe testis from 40 dah. The highest expression level of IGF-3 in testis was at 150 dah. In situ hvbridization analysis showed that IGF-1Ra and-1Rb were expressed in the interstitial cells of both testis and ovary. Additionally, IGF-1Ra and IGF-1Rb were also expressed in spermatocyte. However. No IGF-2R signal was detected in both ovary and testis. Taken together, our data indicated that IGF-IRs, instead of IGF-2R, might be the receptors of IGFs, and IGF-1Ra is more likely to be the receptor of IGF-3.
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