Altered Expression of Protamine-like and Their DNA Binding Induced by Cr(VI): A Possible Risk to Spermatogenesis?

Abstract

Chromium (VI) is the most dangerous oxidation state among the stable forms of chromium. In this work, we evaluated the effect of exposing Mytilus galloprovincialis for 24 h to 1, 10, and 100 nM chromium (VI) on the properties of Protamine-like (PLs) and their gene levels in the gonads. Specifically, we analyzed, by AU-PAGE and SDS-PAGE, PLs extracted from unexposed and exposed mussels. In addition, via EMSA, we evaluated the ability of PLs to bind DNA and also verified their potential to protect DNA from oxidative damage. Finally, we assessed possible alterations in gonadal expression of mt10, hsp70, and genes encoding for PLs-II/PL-IV and PL-III. We found that for all experimental approaches the most relevant alterations occurred after exposure to 1 nM Cr(VI). In particular, a comigration of PL-II with PL-III was observed by SDS-PAGE; and a reduced ability of PLs to bind and protect DNA from oxidative damage was recorded. This dose of chromium (VI) exposure was also the one that produced the greatest alterations in the expression of both mt10 and PL-II/PL-IV encoding genes. All of these changes suggest that this dose of chromium (VI) exposure could affect the reproductive health of Mytilus galloprovincialis.

Keywords: DNA binding; DNA oxidative damage; Mytilus galloprovincialis; chromium; gonad; protamine-like proteins; reproduction; sperm chromatin; spermatozoa; stress and protamine-like proteins genes.

Figure 1

AU-PAGE of PLs extracted from mussels. Lane (1) Chaetopterus variopedatus sperm H1 Histone; lane (2) PL extracted from unexposed mussels (CTR, ie control); lanes (3–5) PL extracted from mussels exposed to 1, 10, and 100 nM chromium (VI), respectively. CTR = unexposed mussels.

Figure 2

SDS-PAGE (panel (a)) and densitometric bands analyses (panel (b)) of PLs extracted from mussels. Lane (1) PLs extracted from unexposed mussels (CTR); Lane (2–4) PLs extracted from mussels exposed to 1, 10 and 100 nM chromium (VI), respectively. CTR = unexposed mussels.

Figure 3

EMSA with: (a) PLs from unexposed mussels; (b) PLs extracted from mussels exposed to 1 nM chromium (VI); (c) PLs extracted from mussels exposed to 10 nM chromium (VI); (d) PLs extracted from mussels exposed to 100 nM chromium (VI). Only plasmid denotes plasmid DNA without proteins; relaxed = relaxed DNA plasmid; supercoiled = supercoiled DNA plasmid.

Figure 4

EMSA with sperm DNA conducted with PLs from unexposed mussels (CTR) and exposed to 1 nM, 10 nM and 100 nM chromium (VI). CTR = unexposed mussels.

Figure 5

Protection assays with PL extracted from: unexposed (CTR) and exposed mussels to 1 nM chromium (VI) (a); exposed to 10 nM and 100 nM chromium (VI) (b). CTR = unexposed mussels.

Figure 6

Protection assay conducted with sperm DNA and PL extracted from unexposed and exposed mussels to 1 nM, 10 nM, and 100 nM chromium (VI). CTR = unexposed mussels.

Figure 7

RT-qPCR of hsp70, mt10, PL-II/PL-IV and PL-III in M. galloprovincialis gonads. In the figure, the change in expression of hsp70 (a), mt10 (b), PL-II/PL-IV (c), and PL-III (d) is reported under the three chromium (VI) exposure conditions compared to the control condition (unexposed mussels). Expression was determined with respect to the housekeeping gene GAPDH. Values are presented as mean ± S.D. (n = 6). Asterisks indicate a statistically significant difference from unexposed mussels: * = p < 0.05; *** = p < 0.001.

Figure 8

ANS-PLs fluorescence analysis. (a) Spectra of ANS fluorescence emission alone (1) and in presence of PLs at (2) 0.01 mg/mL and (3) 0.02 mg/mL. (b) Spectra of ANS fluorescence emission in presence of PLs at increasing concentration of chromium (VI) (1 = 1 nM, 2 = 10 nM, 3 = 100 nM). (c) Plot of the maximum fluorescence intensity values of ANS in presence of PLs at increasing concentration of chromium (VI) in the range from 0 to 5 nM. (d) Plot of the maximum fluorescence emission wavelength of ANS in presence of PLs at increasing concentration of chromium (VI) in the range from 0 to 5 nM. All measurements in (c,d) were performed at least three times at room temperatures.