Spermatozoa Transcriptional Response and Alterations in PL Proteins Properties after Exposure of Mytilus galloprovincialis to Mercury

Abstract

Mercury (Hg) is an environmental pollutant that impacts human and ecosystem health. In our previous works, we reported alterations in the properties of Mytilus galloprovincialis protamine-like (PL) proteins after 24 h of exposure to subtoxic doses of toxic metals such as copper and cadmium. The present work aims to assess the effects of 24 h of exposure to 1, 10, and 100 pM HgCl2 on spermatozoa and PL proteins of Mytilus galloprovincialis. Inductively coupled plasma–mass spectrometry indicated accumulation of this metal in the gonads of exposed mussels. Further, RT-qPCR analyses showed altered expression levels of spermatozoa mt10 and hsp70 genes. In Mytilus galloprovincialis, PL proteins represent the major basic component of sperm chromatin. These proteins, following exposure of mussels to HgCl2, appeared, by SDS-PAGE, partly as aggregates and showed a decreased DNA-binding capacity that rendered them unable to prevent DNA damage, in the presence of CuCl2 and H2O2. These results demonstrate that even these doses of HgCl2 exposure could affect the properties of PL proteins and result in adverse effects on the reproductive system of this organism. These analyses could be useful in developing rapid and efficient chromatin-based genotoxicity assays for pollution biomonitoring programs.

New Insights into Alterations in PL Proteins Affecting Their Binding to DNA after Exposure of Mytilus galloprovincialis to Mercury—A Possible Risk to Sperm Chromatin Structure?

“…The graph describing the results obtained indicated that the absorbance at 420 nm of the PL solution slightly increased as the concentration of HgCl 2 incremented differently from that of Bovine Serum Albumin (BSA) ( Figure 1 a). This result suggests the possible formation of protein aggregates, corroborating that already obtained in Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ( Figure 1 b) [ 28 ], in which additional protein bands with reduced mobility were observed in the samples of PL proteins extracted from mussels exposed to 1, 10 and 100-pM HgCl 2 .…”

“…In order to identify any changes in the PL protein electrophoretic mobility after the exposure of mussels to HgCl 2 , we performed an electrophoretic analysis by SDS-PAGE, maintaining the same final percentage of acrylamide described by Lettieri et al 2021 [ 28 ] but changing the Acrylamide-Bisacrylamide ratio (29:1) in order to obtain an higher resolution of the protein bands. In these conditions, the electrophoretic pattern by SDS-PAGE of the PLII and PLIII proteins ( Figure 3 ) showed that after the exposure of mussels to 10-pM HgCl 2 ( Figure 3 b, lane 3), the band corresponding to the PLII protein did not seem to appear on the gel in the position observed in the control ( Figure 3 b, lane 1).…”

“…Considering the extensive exposure of organisms and the well-recognized toxicity of mercury that causes, even with low-level exposure, several negative impacts on different biological functions, in this work, we deepened our studies on the effects of this metal on the reproductive health of M. galloprovincialis , having already reported not only alterations in the expression of some gonadal stress genes but, also, on the properties of the PL proteins, which represent the main nuclear basic protein component of the sperm chromatin of this organism [ 28 ]. First, we confirmed our hypothesis that mercury could induce PL protein aggregate formation.…”

“…Some hydrophobic residues such as Cys, Ala and Gly undergo much more conformational adaptations at the interface than others [ 35 ], and the two latter amino acids are particularly abundant in PL proteins. As is well-known, mercury is capable of binding cysteines [ 28 ] and histidines [ 36 ]. The binding of mercury ions to these amino acids may underlie the conformational changes we observed from the fluorescence analyses, but we cannot exclude that mercury could also be able to bind other amino acids in which PL proteins are richer, such as glycine and alanine [ 37 ].…”

“…We have previously shown alterations in the properties of protamine-like proteins (PL) of M. galloprovincialis after exposure to sublethal doses of metals such as copper and cadmium [ 3 , 4 , 27 ]. Recently, we showed that the exposure of M. galloprovincialis for 24 h to 1, 10 and 100 pM HgCl 2 caused protamine-like (PL) proteins that were partly in the form of aggregates and were unable to bind and protect DNA from oxidative damage [ 28 , 29 ]. These exposure doses reflected the amount of mercury present in the waters of the Mediterranean basin and the North Atlantic oceans [ 9 , 30 , 31 ].…”