The use of yolk protein as biomarkers for endocrine disruption in molluscs

Invertebrates and especially molluscs have
received increasing attention in relation to endocrine disrupting
chemicals (EDs) during the last few years and the development of
OECD test guidelines to assess the effect of EDs with molluscs are in
progress. One of the main problems with the development of
standardized tests in molluscs is that no specific biomarkers or endpoints
for endocrine effects have been validated. Some attempts have been
made to transfer biomarkers developed for vertebrates – e.g. from fish to
molluscs to investigate ED effects. One example is the vertebrate yolk
protein vitellogenin that is known to be oestrogen dependent in fish. The
yolk proteins in molluscs have been proposed to have the same
oestrogenic dependence and used as biomarker for oestrogenic EDs. The
present work investigates the possible usability of the main yolk protein
in three species of molluscs to function as biomarker for oestrogenic
exposure. We have developed specific antibodies against the main yolk
protein from the three species (Unio pictorum, Unio tumidus and
Lymnaea stagnalis) and with specific ELISAs (enzyme linked immunesorbent
assay) and immune-histology we investigate the distribution and
concentration of the proteins in molluscs of different sex and life-stage.
The main yolk protein was purified from gonads containing eggs of the
freshwater bivalves U. pictorum and U. tumidus and from eggs dissected
from egg clutches of the pulmonate gastropod L. stagnalis. The ELISAs
were used to quantify the concentration of the yolk protein in juveniles
and adult male and female U. pictorum and U. tumidus and in the
hermaphroditic L. stagnalis. With use of immune-histology, the
distribution of the proteins in the tissue of the three species was
investigated. The ELISAs revealed that the normal sex specific
concentration distribution seen in fish, where vitellogenin is normally
seen in very low concentrations in male and juveniles was not seen in
the molluscs. The concentration of the protein did not differ among the
sexes and was a factor of approximately 10000 higher in male U.
tumidus and U. Pictorum than in male fish. Based on The results the
authors do not support the use of mollusc yolk protein as biomarker for
oestrogenic exposure because it seems to have more than one function in
the investigated species. We have shown that the content and
distribution of the proteins are not sex specific.