Alterations in the properties of sperm protamine-like II protein after exposure of Mytilus galloprovincialis (Lamarck 1819) to sub-toxic doses of cadmium.

Protamine-like proteins (PL-II, PL-III and PL-IV) represent the major basic nuclear component of Mytilus galloprovincialis L sperm chromatin. The present study investigates the effects induced on the properties of PL-II protein after exposure of Mytilus galloprovincialis L for 24 h to 1.5 and 5 µM CdCl2. We found cadmium accumulation in protamine-like proteins with a linear grow up with the exposition dose. In particular, after 5 µM CdCl2 mussels exposure, the mobility of PL-II band changed in SDS-PAGE, suggesting structural rearrangement in presence of cadmium. Structural analysis using fluorescent probes, indicated that at 5 µM CdCl2 the complete conformational change of PL-II protein was reached. In the same condition of mussels exposure of 5 µM CdCl2, PL-II protein changed its DNA binding mode, which determined a closer DNA binding, because higher amount of NaCl were required for PL-II protein release by sperm nuclei. These results supported the hypothesis that mussel exposure to this CdCl2 dose, although lower to toxic ones, affects the properties of this protein and as a consequence chromatin organization of spermatozoa that is essential for the success of fertilization.

Protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on male mollusk Mytilus galloprovincialis (Lamarck 1819).

Here we report the industrial pollution effects due to cadmium on the reproductive health of Mytilus galloprovincialis. Mussels were removed from the biofouling of a Conatex panel after one year exposition at a polluted site near a disposal metallurgical factory. A high cadmium bioaccumulation was observed in the testis of mussels housed at the polluted site, with respect to a control site, as determined by inductively coupled plasma-mass spectrometry, along with a 10 fold increase in metallothionein 20 kDa gene (mt20) expression levels determined by qPCR. Furthermore, mussels transferred into laboratory tanks from the reference site, and exposed to 1.5, 5 and 10 µM CdCl2, revealed a 1.7, 3.2 and 4.5 fold expression increase in the testis mt20, respectively, and a positive correlation with cadmium bioaccumulation was found. To evaluate a potential detrimental risk of such alterations on spermatozoa, we carried out electrophoretic analyses on their protamine-like proteins. As determined by AU-PAGE, after 1.5 µM CdCl2 exposure, protamine-like proteins also display major alterations with respect to those obtained after 5 and 10 µM CdCl2 exposure. All protamine-like proteins isolated from the polluted biofouling were in an aggregated form and displayed the same reduced DNA binding affinity of the protamine-like proteins obtained after 1.5 µM CdCl2 as demonstrated EMSA with sperm genomic DNA. Our results contribute to the studies concerning cadmium induced testis alterations and highlight protamine-like proteins' analysis as an emerging biotechnique for cadmium impact assessment on Mytilus galloprovincialis, for the sensitivity of the in vivo and in vitro changes of protamine-like proteins' state and their DNA binding affinity.