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?

Abstract

Mercury (Hg) is a highly toxic and widespread pollutant. We previously reported that the exposure of Mytilus galloprovincialis for 24 h to doses of HgCl₂ similar to those found in seawater (range 1-100 pM) produced alterations in the properties of protamine-like (PL) proteins that rendered them unable to bind and protect DNA from oxidative damage. In the present work, to deepen our studies, we analyzed PL proteins by turbidimetry and fluorescence spectroscopy and performed salt-induced release analyses of these proteins from sperm nuclei after the exposure of mussels to HgCl2 at the same doses. Turbidity assays indicated that mercury, at these doses, induced PL protein aggregates, whereas fluorescence spectroscopy measurements showed mercury-induced conformational changes. Indeed, the mobility of the PLII band changed in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, particularly after exposure to 10-pM HgCl₂, confirming the mercury-induced structural rearrangement. Finally, exposure to HgCl₂ at all doses produced alterations in PL-DNA binding, detectable by DNA absorption spectra after the PL protein addition and by a decreased release of PLII and PLIII from the sperm nuclei. In conclusion, in this paper, we reported Hg-induced PL protein alterations that could adversely affect mussel reproductive activity, providing an insight into the molecular mechanism of Hg-related infertility.

Keywords: HgCl2; PL proteins; fluorescence spectroscopy; mussel; reproduction; sperm nuclei; spermatozoa; turbidity assays.

Figure 1

Turbidity assay (a) following 420-nm absorbance changes as a function of HgCl₂ adding: in black, the trend of PL proteins, and, in red, that of BSA (negative control). An electrophoretic analysis by SDS PAGE (b) of M. galloprovincialis PL proteins extracted from nonexposed (lane 1) and exposed mussels (lanes 2, 3 and 4): to 1, 10 and 100-pM HgCl₂, respectively. (b) A reused figure that corresponds to (B) of Figure 3 (Lettieri et al. 2021) obtained under the Creative Common CC BY license. Lettieri G, Notariale R, Ambrosino A, Di Bonito A, Giarra A, Trifuoggi M, Manna C, Piscopo M. Spermatozoa Transcriptional Response and Alterations in PL Proteins Properties after Exposure of M. galloprovincialis to Mercury. Int J Mol Sci. 2021 Feb 5;22(4):1618. doi: 10.3390/ijms22041618.

Figure 2

Fluorescence spectra of PL proteins from nonexposed (control) and HgCl₂-exposed mussels.

Figure 3

SDS-PAGE (b) and densitometric bands analyses (c) of the PLII and PLIII proteins, extracted from spermatozoa of nonexposed mussels (lane 1) and of mussels exposed to 1, 10 and 100-pM HgCl₂ (lanes 2, 3 and 4, respectively). AU-PAGE (a) of the PL proteins from the spermatozoa of nonexposed mussels (lane 1) and of mussels exposed to 1, 10 and 100-pM HgCl₂ (lanes 2, 3 and 4, respectively) (n = 6).

Figure 4

Absorption spectra in the range of 200–300 nm of plasmid DNA alone and PL/DNA mixtures at various PL/DNA ratios: PL extracted from nonexposed mussels (a) and PL extracted from mussels exposed to 1 pM (b), 10 pM (c) and 100-pM HgCl₂ (d), respectively. Green: plasmid alone, yellow: PL/DNA 0.5 ratio (w/w), blue: PL/DNA ratio 1 (w/w), grey: PL/DNA ratio 1.5 (w/w), blue: PL/DNA ratio 2 (w/w) and red: PL/DNA ratio 3 (w/w).

Figure 5

The release of PL from sperm nuclei at different NaCl molar concentrations in the control mussels and exposed to 1, 10 and 100-pM HgCl₂ (n = 6).