alpha2-Macroglobulin in the marine fish Sparus aurata.

The alpha2-macroglobulin proteinase inhibitors (alpha2Ms) are a family of plasma proteins with the unique ability to inhibit a broad spectrum of proteinases, but are also known as binding proteins for many growth factors and cytokines, including growth hormone and members of the transforming growth factor-beta superfamily. A partial cDNA (475 amino acids) encoding the C-terminus of alpha2M was cloned from the liver of the marine teleostean fish Sparus aurata. The deduced amino acid sequence of the cloned fragment showed 58-60% similarity to carp alpha2Ms. Northern blot analysis of hepatic alpha2M revealed a transcript of about 5 kb. A transcript of a similar size was detected in 1-day larvae. Steady state levels of alpha2M in larvae increased gradually on subsequent days post-hatching. alpha2M expression in embryos was determined by RT-PCR and started in embryos aged 8 h post-fertilization, but not earlier. RT-PCR of muscle RNA detected alpha2M also in fish muscle, albeit with a lower expression than in the liver. Immunoreactive-alpha2M was found in yolk syncytial layer of 3-day larvae and in livers from larvae and adults. Immunoreactive-alpha2M was also identified in soluble total proteins from young larvae with a pattern resembling that of plasma. These data demonstrate that the alpha2M gene is expressed early in fish development. Moreover, in addition to its major expression in liver, alpha2M is expressed also in fish muscle.

Application of real-time PCR for quantitative determination of hepatic vitellogenin transcript levels in the striped sea bream, Lithognathus mormyrus.

The striped sea bream (Lithognathus mormyrus) has been recently introduced as a bioindicator fish species in Mediterranean coastal habitats. The purpose of this study was to apply a real-time PCR assay for the determination of absolute levels of hepatic VTG transcript in this fish, identifying minimal and maximal levels and establishing the relationship between VTG RNA levels and ovarian development. A partial VTG cDNA was cloned from hepatic RNA of a striped sea bream female. Specific primers were designed based on its sequence and used for PCR and also for in vitro synthesis of partial VTG RNA standard. Hepatic VTG transcript levels were quantified by real-time PCR, using serially diluted VTG RNA standards for construction of a calibration curve and equation. VTG RNA levels were normalized to total RNA or 18S ribosomal RNA (determined by real-time PCR). VTG RNA was hardly detected in the liver of males, or females with small oocytes (diameter < 100 microm). A linear correlation was found between these two parameters at larger oocyte diameter (> 150 microm). VTG level reached a maximum of 204 fmol/pmol 18S RNA or 49 fmol/microg RNA. The results demonstrate the wide dynamic range of the established real-time PCR assay.