A Molecular Mechanism to Explain the Nickel-Induced Changes in Protamine-like Proteins and Their DNA Binding Affecting Sperm Chromatin in Mytilus galloprovincialis: An In Vitro Study

Abstract

Nickel is associated with reproductive toxicity, but little is known about the molecular mechanisms of nickel-induced effects on sperm chromatin and protamine-like proteins (PLs). In the present work, we analyzed PLs from Mytilus galloprovincialis by urea-acetic acid polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and assessed their binding to DNA by Electrophoretic Mobility Shift Assay (EMSA) after exposing mussels to 5, 15, and 35 µM NiCl₂ for 24 h. In addition, a time course of digestion with MNase and release of PLs from sperm nuclei by the NaCl gradient was performed. For all exposure doses, in AU-PAGE, there was an additional migrating band between PL-III and PL-IV, corresponding to a fraction of PLs in the form of peptides detected by SDS-PAGE. Alterations in DNA binding of PLs were observed by EMSA after exposure to 5 and 15 µM NiCl₂, while, at all NiCl₂ doses, increased accessibility of MNase to sperm chromatin was found. The latter was particularly relevant at 15 µM NiCl₂, a dose at which increased release of PLII and PLIII from sperm nuclei and the highest value of nickel accumulated in the gonads were also found. Finally, at all exposure doses, there was also an increase in PARP expression, but especially at 5 µM NiCl₂. A possible molecular mechanism for the toxic reproductive effects of nickel in Mytilus galloprovincialis is discussed.

Keywords: DNA; Mytilus galloprovincialis; PARP; nickel; nickel molecular mechanisms; protamine-like; sperm chromatin; spermatozoa.

Figure 1

Evaluation of nickel bioaccumulation in gonad. n = 3. * = p < 0.05.

Figure 2

AU-PAGE (a) and SDS-PAGE (b) analyses of M. galloprovincialis PL proteins extracted from unexposed (Unx) (lane 2 of panels (a,b)) and exposed mussels (lanes 3–5 of panels (a,b)) to 5, 15, and 35 µM NiCl₂, respectively. Lane 1 of AU-PAGE shows sperm H1 histone from the annelid worm Chaetopterus variopedatus. M in SDS-PAGE (panel (b), lanes 1 and 6) denotes molecular weight marker. (c) evaluation of the % of intermediate band respect to PLs, by densitometric analysis. The intermediate band is the additional protein band migrating between PL-III and PL-IV, visible in AU-PAGE. n = 3.

Figure 3

Analysis of the ability of PL proteins to bind DNA from unexposed (a) and exposed mussels to 5, 15, and 35 µM NiCl₂ ((b), (c), and (d), respectively) by EMSA, using pGEM3 plasmid DNA. Numbers on the wells indicate PL protein/DNA (w/w) ratios used; only DNA indicates pGEM3 plasmid DNA without added protein. n = 3. Relaxed and supercoiled are the two topological forms of pGEM3 plasmid DNA.

Figure 4

Release of PL proteins from sperm nuclei with increasing molar concentrations of NaCl in the unexposed mussels and exposed to 5, 15, and 35 µM NiCl₂. The data are presented as mean ± S.D. n = 3. (a): PL-II; (b): PL-III and (c): PL-IV.

Figure 5

MNase digestion time course of M. galloprovincialis sperm chromatin. Analyses of DNA by electrophoresis on 0.9% agarose gel: unexposed (panel (a)) and exposed mussels to 5, 15, and 35 µM (panels (b), (c), and (d), respectively). n.d. = Sperm DNA not digested; n = 3.

Figure 6

Western blotting carried out in homogenates of M. galloprovincialis gonads of unexposed and exposed mussels to 5, 15, and 35 µM NiCl₂. (a): 12% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (b): anti-PARP immunoblotting. (c,d): densitometric analysis. The histograms represent mean ± SD. Results were analyzed by Kruskal–Wallis’ test: the intensity of 50 kDa band (a) and that between 30–40 kDa (b) in samples of exposed mussels to 5 and 15 µM NiCl₂ was significantly higher than that determined in the unexposed condition. n = 3.

Figure 7

Hypothetical molecular model that explains nickel-induced reproductive toxicity in M. galloprovincialis.