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

Abstract

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 CdCl, 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 CdCl exposure, protamine-like proteins also display major alterations with respect to those obtained after 5 and 10 µM CdCl 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 CdCl 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.

Molecular effects on spermatozoa of Mytilus galloprovincialisexposed to hyposaline conditions

“…4.4 | PL proteins extraction from M. galloprovincialis spermatozoa M. galloprovincialis PL proteins from spermatozoa were extracted using 5% perchloric acid (PCA) as previously described (Piscopo, Trifuoggi, Notariale, et al, 2018). In summary, after the homogenization of 10 spermatozoa pellets (for each saline condition) in 15 ml of distilled water at 4°C, we added PCA to a final concentration of 5%.…”

“…Further, we have recently proposed the PL proteins both as antibacterial agents (Notariale et al, ) and as an emerging biomarker for cadmium impact assessment on male M . galloprovincialis (De Guglielmo et al, ; Piscopo, Notariale, et al, ; Piscopo, Notariale, & Troisi, ; Piscopo, Trifuoggi, Notariale, et al, ).…”

“…They represent the 76% of the overall nuclear protein in M. galloprovincialis with PL-III (11.3 kDa) present in the highest amount (50%) and PL-II (14 kDa), and PL-IV (6.5 kDa), which represent the 20% and 6%, respectively (Vassalli et al, 2015). Further, we have recently proposed the PL proteins both as antibacterial agents and as an emerging biomarker for cadmium impact assessment on male M. galloprovincialis (De Guglielmo et al, 2019;Piscopo, Notariale, & Troisi, 2017;Piscopo, Trifuoggi, Notariale, et al, 2018).…”

Toxicity assessment of nano‐TiO2inApis melliferaL., 1758: histological and immunohistochemical assays

“…These biomarkers, used in previous studies, have proved excellent biomarkers of exposure to metallic xenobiotic in different animals (Droutsa et al, ; Ferrante et al, ; Mazzei et al, ; Piscopo et al, ; Salvaggio et al, ); they have been chosen because it has been shown that they intervene in the detoxification processes of metallic nanoparticles (Brundo et al, ; Pecoraro, Marino, et al, ; Pecoraro, Salvaggio, et al, ).…”

Protamine-like proteins have bactericidal activity. The first evidence in Mytilus galloprovincialis

“…Several bioactive compounds have been identified in marine organisms which represent a large reservoir for pharmacologically potent and active drugs that evolved to deal with extremely difficult, competitive, and aggressive constraints of the environment in which they live (Piscopo et al, 2018b;Maresca et al, 2018), and which are very different compared to those in terrestrial environments (Conte et al, 2017). Cationic compounds have been demonstrated as potential candidates for new bactericidal agents since they have a low potential for the development of resistance (Carmona-Ribeiro et al, 2013).…”