Enzyme-linked immunosorbent assay (ELISA) measurement of vitellogenin in plasma and liver histopathology in barfin plaice Liopsetta pinnifasciata from Amursky Bay, Sea of Japan

Abstract

Vitellogenin (Vg) of the barfin plaice Liopsetta pinnifasciata was isolated and purified. In native polyacrylamide gel electrophoresis, Vg appeared as one band. After being subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Vg fraction produced several polypeptides with molecular masses of 180, 98, 70, 52, 41 and 37 kDa. MALDI-TOF mass spectrometry (MS) of the 180- and 98-kDa Vg polypeptides from the SDS-PAGE gel and de novo sequencing of their four peptide fragments based on MS/MS analysis confirmed that the purified proteins were vitellogenins, which shared high similarity with the Vgs of the barfin flounder Verasper moseri and Atlantic halibut Hippoglossus hippoglossus. The most part of the predicted sequences obtained from the L. pinnifasciata 180-kDa polypeptide has previously been found in the V. moseri vitellogenin type B, the sequences obtained from the 98-kDa polypeptide were found in V. moseri vitellogenin type A, so these findings allow us to propose that L. pinnifasciata has at least two different forms of Vg. Rabbit polyclonal antibodies against Vg were produced, and a quantitative enzyme-linked immunosorbent assay was developed. The concentration of Vg in barfin plaice from the moderately contaminated area of Amursky Bay in the Sea of Japan was detected based on the maturity stage of their gonads. In November 2008, the Vg concentration in the plasma of females with advanced oogenesis varied from 5.295 to 28.367 mg/ml (mean 16.38 ± 6.73 mg/ml, CV = 41.1%); in the plasma of males, the concentration ranged from non-detectable to 0.957 mg/ml (0.29 ± 0.42 mg/ml, CV = 127.9%). In October 2009, the Vg concentration in female plasma was lower than in November 2008 (2.21-13.87 mg/ml). High individual variability of plasma Vg was characteristic for maturing males (CV = 200.3%) and immature females (CV = 255.5%), and there was no significant difference between plasma Vg concentrations in males captured in November 2008 and October 2009 or in maturing males and immature females. Vacuolisation of hepatocytes was more typical for males with low plasma Vg concentrations and females with high plasma Vg concentrations. Necrosis and pyknosis of hepatocyte nuclei were more frequent in males with high Vg concentrations and in females with low plasma Vg concentrations.

Hexestrol- and nonylphenol-induced differential vitellogenin synthesis in female and male barfin plaice Liopsetta pinnifasciata

“…4). The significant increase in concentration of the 98-kDa polypeptide corresponds to an increase in VtgAa levels according to our previous results (Shved et al, 2011).…”

“…1b). The significant increase in concentration of the 180-kDa polypeptide corresponds to an increase in VtgAb levels according to our previous results (Shved et al, 2011). In experimental fish, the increase in quantity of polypeptide with molecular mass of 98 kDa corresponding to VtgAa type wasn't noted.…”

“…From this point of view barfin plaice Liopsetta pinnifasciata is poorly studied species. In previous our paper (Shved et al, 2011) we found two types of Vtgs in L. pinnifasciata that shared high similarity with Aa and Ab types of the barfin flounder Verasper moseri and Atlantic halibut Hippoglossus hippoglossus. When subjected to native electrophoresis, the Vtgs behaved identically and exhibited a single band on the gel.…”

“…For the autumn exposed group, SDS-PAGE analysis indicated that accumulation of Vtg in the plasma was due to a preferential accumulation of protein with a molecular mass of 98 kDa. In our previous paper (Shved et al, 2011), we illustrated via MALDI-TOF that this polypeptide has a high similarity with the A-type Vtg of barfin flounder V. moseri. Consequently, the induced Vtg can be classified as VtgA according to classification of Hiramatsu et al (2006) or VtgAa as in the classification of Finn and Kristofferson (2007).…”

“…A detailed description of the method was published in our previous paper (Shved et al, 2011). PAbs were purified as the IgG fraction.…”