The Immune Function Of Vitellogenin From Zebrafish Danio Rerio

Vitellogenin (Vg), a phospholipoglycoprotein, is the precursor of major egg-yolk proteins in all oviparous species from invertebrates to vertebrates. All forms of fish Vgs are synthesized by the liver of females in response to the endogenous estrogen, 17-β-estradiol (E2), secreted into bloodstream and transported to ovary, where they are internalized by growing oocytes via receptor-mediated endocytosis and proteolytically cleaved to form the egg-yolk proteins that are later used as the nutrients by developing embryos and larvae. Normally, Vgs are undetectable in male and immature female fish; however, their synthesis can be induced by exogenous E2. On the other hand, synthesis of both Vg mRNA and proteins can be inhibited by tamoxifen (TMX), a E2 antagonist. Recent studies have shown that in addition to being involved in egg-yolk protein formation, Vg appears to have evolved pleiotropic functions. A novel function of Vg is linked with immune defense. In the protochordate amphioxus (Branchiostoma belcheri) as well as the bony fish rosy barb (Puntius conchonius), Vgs have been demonstrated to possess both hemagglutinating and antibacterial activities. In vitro experiments have shown that fish Vg acts as a multivalent pattern recognition receptor with an opsonic activity. However, the potential immunologic role of vitellogenin in vivo remains obscure. This study demonstrated that serum Vg in zebrafish D. rerio is an acute phase protein with bacterial-binding and inhibiting activities. It also bolsters the notion that factors normally involved in control of female reproduction are linked with immunity in organisms that rely on Vg for oocyte development. This study also indicated that Vg may enhance the survival rates of D. rerio following challenge with V. anguillarum.This paper examined whether the injection of pathogen-associated molecular patterns (LPS and LTA) to male D. rerio could trigger Vg synthesis in vivo. Male D. rerio were injected with LPS, LTA or 0.9% NaCl solution (control) and were sampled at 0h, 0.5h, 1h, 2h, 3h, 5h, 9h and 12h post injection (hpi) for total RNAs and serums. Real-time PCR showed that LPS could trigger Vg1 gene expression in vivo immediately after injection. Vg1 gene expression of LPS-induced male D. rerio was about 104.1-fold of the control at 0.5 hpi, reached the peak of about 193.2-fold at 2 hpi and kept in high levels at the following time points. Vg1 gene expression induced by LTA increased to 45-fold at 0.5 hpi but quickly decreased to the control level at the following time points. The results of ELISA showed that serum Vg levels increased significantly to 1.3-fold of the control at 0.5 hpi after injection with LPS while it increased to 1.2-fold at 9 hpi after injection with LTA. It revealed that LPS as well as LTA could significantly trigger Vg synthesis in vivo in both mRNA and protein levels in male D. rerio. The response of Vg induced by PAMPs revealed that Vg was an acute phase protein. We applied Western blotting to detect whether Vg can bind to E.coli and S.aureus. The serum of LPS-induced male D. rerio at 0.5 hpi was incubated with E.coli and S.aureus respectively. Then the protein binding to bacteria were eluted carefully. The elution was carried out for western blotting with rabbit anti-Vg polyconal antibody. The results showed that Vg was capable of binding to both E.coli and S.aureus. Inhibition of bacterial growth by serum Vg induced by LPS was also detected. The sera from control and LPS-injected male D. rerio all exhibited conspicuous antibacterial activities against E. coli and S. aureus, with the antibacterial activities of the sera collected at 0.5 hpi from LPS-injected fish being significantly higher than those from saline-injected fish. When the complement activities of the sera were inactivated by heating, their antibacterial activities against E. coli and S. aureus were remarkably reduced. However, some antibacterial activities remained in all the heated sera. In particular, after heating, the antibacterial activities of the sera collected at 0.5 hpi from LPS-injected fish were still significantly higher than control, suggesting the presence of additional antibacterial molecules in these sera. Notably, the antibacterial activities of the heated sera against E. coli and S. aureus were significantly inhibited by the pre-incubation of the sera with anti-zebrafish Vg antibody, but not by the pre-incubation with anti-actin antibody; and this inhibitory effect showed a clear dose-dependence. Moreover, consistent with higher Vg levels in the sera collected at 0.5 hpi, the antibacterial activities in these sera, after heating, were also greater than control. These data together implicated that Vg was an important factor responsible for the antibacterial activities in the sera.In the second part, prior experiments were firstly applied to determine the appropriate concentrations and time of the exposure. The results showed that Vg synthesis in D. rerio exposed to 20 nM E2 at 20 dpe (days after exposure) was significantly up-regulated while it was significantly down-regulated by 500 nM TMX at 20dpe. However, histological examination revealed that exposure to 20 nM E2 and 500 nM TMX cause only slight hepatocytic changes in D. rerio. Western blotting showed that both E2 and TMX were able to significantly trigger IgM synthesis in D. rerio. However, no marked difference of IgM levels was detected between E2- and TMX-exposed fish. We also found that exposures to E2 and TMX had little influence on the density of leucocytes in zebrafish blood. Finally, after exposure to 20 nM E2 or 500 nM TMX, for consecutive 20 days, the exposed fish as well as normal fish were injected intraperitonealy with Vibrio auguillarum (V . auguillarum) and the survival rates were calculated at different time points, respectively. The results showed that the survival rates of the E2-exposed females were significantly higher than those of normal females and TMX-exposed females. In males, E2-exposed group exhibited significantly higher survival rates than TMX-exposed group but similar to those of normal group. The results indicated that Vg may improve the survival rates of D. rerio following challenge with V. auguillarum.In summary, this paper revealed that Vg in zebrafish D. rerio is an acute phase protein with bacterial-binding and inhibiting activities. The PAMPs (LPS and LTA) can induce Vg synthesis in vivo in male D. rerio, and the response of Vg revealed an acute-phase response. Serum Vg induced by PAMPs showed antibacterial activities to both E.coli and S.aureus. In vivo experiments also showed that, Vg may improve the survival rates of D. rerio following challenge with V. auguillarum.