Isolation, purification and characterization of the egg-yolk proteins from the oocytes of the indian freshwater murrel, Channa punctatus (Bloch).

In oviparous organisms, yolk accumulation in the oocytes is critical and indispensable for the development of the newly hatched young ones. In fish and many other oviparous vertebrates, the major constituents of the egg-yolk are synthesized as a precursor in the liver. The precursor is transported to the oocyte for uptake and cleaved into major yolk proteins lipovitellin, phosvitin and beta'-components. The eggs of Channa punctatus are pelagic, have large oil globule and exceptionally high lipid content. Lipovitellin was isolated by single step gel filtration chromatography on Sepharose 6B. Purified native lipovitellin showed immunological reactivity with vitellogenin antiserum. Phosvitin isolated by phenol extraction method could not be visualized with routine protein staining methods, whereas incorporation of trivalent ions in the coomassie brilliant blue stained phosvitin. It was characterized by in vivo labeling of egg-yolk proteins with 32P. The molecular mass of murrel phosvitin was less than 14,000 kDa.

Vitellogenin genes in fish: differential expression on exposure to estradiol.

Three types of vitellogenins (Vgs) namely vitellogenin A (VgA), vitellogenin B (VgB) and vitellogenin C (VgC) have been identified in fishes. The existence of VgA and VgB is reported in the Indian freshwater murrel Channa punctatus. Gene-specific primers were designed using available nucleotide sequences in National Centre for Biotechnology Information (NCBI), for amplification of VgA and VgB cDNA. Differential processing of Vgs is evident in many fishes. Adult male murrel expressed both the VgA and VgB genes when estradiol-17ß (E(2)) is injected in vivo and Vg levels in blood quantified by Enzyme linked immunosorbent assay (ELISA) showed a dose-related response in such treatments. Cultured hepatocytes on treatment with E(2), however, expressed only VgB as detected by RT-PCR, suggesting different regulatory mechanism for the VgA and VgB genes.

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, Channa punctata: in vivo and in vitro studies.

The relative efficacies of three natural estrogens viz., estrone (E(1)), estradiol-17beta (E(2)) and estriol (E(3)) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001-10 microM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E(1) was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 microM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E(2) or E(3) even at a concentration of 0.001 microM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.