Isolation and characterization of immunoglobulin from African catfish, Clarias gariepinus (Burchell, 1822)

Immunoglobulins are an important aspect of adaptive immune system and tetrameric IgM are the most prevalent immunoglobulins in Pisces.. In this study, we made an attempt to isolate and characterize immunoglobulins from the Afrcian cat fish, Clarias gariepinus (Burchell, 1822). The immunoglobulins were induced by immunization with BSA. Various methods such as ammonium sulphate precipitation, ion exchange chromatography (DEAE cellulose), gel permeation chromatography and affinity chromatography (Protein A & CNBr-activated agarose conjugated with BSA) were employed for purification of immunoglobulins. But for affinity chromatography involving BSA conjugated agarose, all other methods could purify immunoglobulins only partially, i.e., there was contamination of other proteins. Whereas with affinity chromatography, immunoglobulins could be isolated in purified form. Electrophoresis under denaturing condition resulted in one heavy and two light chain bands of molecular weights of 74.5 and 29.7 & 30.5 kDa, respectively. It resolved into single band on electrophoresis under native conditions. The molecular weight of immunoglobulin was estimated to be 890 kDa by gel filtration chromatography on Ultrogel AcA34. The immunoglobulin was further characterized by western blotting and MALDI-MS and N-terminal analysis. Rat anti-fish Ig generated against heavy chain showed cross reactivity with fish antibody raised against BSA or Aeromonas hydrophila.

Aromatase activity in brain and ovary: Seasonal variations correlated with circannual gonadal cycle in the catfish, Heteropneustes fossilis.

Seasonal variations in the aromatase activity in H. fossilis estimated by a microassay were correlated with the sex steroids, vitellogenin in and ovarian weight during circannual reproductive cycle. In the female catfish, aromatase activity was detectable in the hypothalamus throughout the year whereas in ovary only during active vitellogenesis. In the catfish, hypothalamic aromatase levels increased two times during annual gonadal cycle, once in a fully gravid fish and then in a reproductively quiescent fish. On the other hand, increase in the ovarian aromatase activity was observed only during vitellogenesis, which showed a direct correlation with plasma levels of sex steroids. Further, plasma levels of testosterone and estradiol suggested a precursor-product relationship. At the completion of vitellogenesis, ovarian aromatase activity declined sharply resulting in elevation of plasma testosterone levels, which in turn could be utilized as substrate by the hypothalamic aromatase whose activity was the highest in the postvitellogenic catfish. At least two isoforms of gene, cyp19a and cyp19b, coding for aromatase in ovary and brain respectively were expressed in the catfish. Aromatase activity was more concentrated in those areas of catfish brain, which have been implicated in the control of reproduction.

Immunological identification of two female-specific proteins from the plasma of Indian freshwater murrel, Channa punctatus (Bloch).

Existence of a non-phosphorylated female-specific protein (FS II), in addition to phosphorylated vitellogenin (FS I), in the plasma of murrel by exogenous administration of estradiol-17beta is reported. Polyspecific rabbit antibodies were raised against estrogen-inducible murrel plasma proteins. This antiserum was absorbed with normal male serum in order to obtain female-specific antiserum (FSAS). Radial immunodiffusion studies suggested that both the proteins (FS I and FS II) were present in the plasma of E2-treated and normal vitellogenic females and in the ovarian homogenate from gravid females, but absent in normal male plasma. Autoradiographic experiments demonstrated that phosphorus moiety was attached with FS I only. Further, immunoelectrophoretic analysis and peptide maps supported the observation that FS I and FS II were discrete, unrelated female-specific proteins.