Small noncoding RNAs and sperm nuclear basic proteins reflect the environmental impact on germ cells.

BACKGROUND: Molecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early alterations due to environmental pollution. METHODS: Here, we present a multilevel molecular profiling by small RNA sequencing and sperm nuclear basic protein analysis of male germ cells from 33 healthy young subjects residing in low and high-polluted areas. RESULTS: Although sperm motility and sperm concentration were comparable between samples from the two sites, those from the high-pollution area had a higher concentration of immature/immune cells, a lower protamine/histone ratio, a reduced ability of sperm nuclear basic proteins to protect DNA from oxidative damage, and an altered copper/zinc ratio in sperm. Sperm levels of 32 microRNAs involved in intraflagellar transport, oxidative stress response, and spermatogenesis were different between the two areas. In parallel, a decrease of Piwi-interacting RNA levels was observed in samples from the high-polluted area. CONCLUSIONS: This comprehensive analysis provides new insights into pollution-driven epigenetic alterations in sperm not detectable by spermiogram.

A Metabolomic Analysis to Assess the Responses of the Male Gonads of Mytilus galloprovincialis after Heavy Metal Exposure.

In recent years, metabolomics has become a valuable new resource in environmental monitoring programs based on the use of bio-indicators such as Mytilus galloprovincialis. The reproductive system is extremely susceptible to the effects of environmental pollutants, and in a previous paper, we showed metabolomic alterations in mussel spermatozoa exposed to metal chlorides of copper, nickel, and cadmium, and the mixture with these metals. In order to obtain a better overview, in the present work, we evaluated the metabolic changes in the male gonad under the same experimental conditions used in the previous work, using a metabolomic approach based on GC-MS analysis. A total of 248 endogenous metabolites were identified in the male gonads of mussels. Statistical analyses of the data, including partial least squares discriminant analysis, enabled the identification of key metabolites through the use of variable importance in projection scores. Furthermore, a metabolite enrichment analysis revealed complex and significant interactions within different metabolic pathways and between different metabolites. Particularly significant were the results on pyruvate metabolism, glycolysis, and gluconeogenesis, and glyoxylate and dicarboxylate metabolism, which highlighted the complex and interconnected nature of these biochemical processes in mussel gonads. Overall, these results add new information to the understanding of how certain pollutants may affect specific physiological functions of mussel gonads.

Impact of Heavy Metal Exposure on Mytilus galloprovincialis Spermatozoa: A Metabolomic Investigation.

Metabolomics is a method that provides an overview of the physiological and cellular state of a specific organism or tissue. This method is particularly useful for studying the influence the environment can have on organisms, especially those used as bio-indicators, e.g., Mytilus galloprovincialis. Nevertheless, a scarcity of data on the complete metabolic baseline of mussel tissues still exists, but more importantly, the effect of mussel exposure to certain heavy metals on spermatozoa is unknown, also considering that, in recent years, the reproductive system has proved to be very sensitive to the effects of environmental pollutants. In order to fill this knowledge gap, the similarities and differences in the metabolic profile of spermatozoa of mussels exposed to metallic chlorides of copper, nickel, and cadmium, and to the mixture to these metals, were studied using a metabolomics approach based on GC-MS analysis, and their physiological role was discussed. A total of 237 endogenous metabolites were identified in the spermatozoa of these mussel. The data underwent preprocessing steps and were analyzed using statistical methods such as PLS-DA. The results showed effective class separation and identified key metabolites through the VIP scores. Heatmaps and cluster analysis further evaluated the metabolites. The metabolite-set enrichment analysis revealed complex interactions within metabolic pathways and metabolites, especially involving glucose and central carbon metabolism and oxidative stress metabolism. Overall, the results of this study are useful to better understand how some pollutants can affect the specific physiological functions of the spermatozoa of this mussel, as well as for further GC-MS-based metabolomic health and safety studies of marine bivalves.

Possible Molecular Mechanisms Underlying the Decrease in the Antibacterial Activity of Protamine-like Proteins after Exposure of Mytilus galloprovincialis to Chromium and Mercury.

Natural bioactive compounds represent a new frontier of antimicrobial molecules, and the marine ecosystem represents a new challenge in this regard. In the present work, we evaluated the possibility of changes in the antibacterial activity of protamine-like (PL) proteins, the major nuclear basic protein components of Mytilus galloprovincialis sperm chromatin, after the exposure of mussels to subtoxic doses of chromium (VI) (1, 10, and 100 nM) and mercury (1, 10, and 100 pM) HgCl2, since these metals affect some properties of PL. After exposure, we analyzed the electrophoretic pattern of PLs by both acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and SDS-PAGE and determined the MIC and MBC of these proteins on different gram+ and gram- bacteria. PLs, particularly after mussels were exposed to the highest doses of chromium and mercury, showed significantly reduced antibacterial activity. Just at the highest doses of exposure to the two metals, changes were found in the electrophoretic pattern of PLs, suggesting that there were conformational changes in these proteins, which were confirmed by the fluorescence measurements of PLs. These results provide the first evidence of a reduction in the antibacterial activity of these proteins following the exposure of mussels to these metals. Based on the results, hypothetical molecular mechanisms that could explain the decrease in the antibacterial activity of PLs are discussed.

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.

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.

Molecular Alterations and Severe Abnormalities in Spermatozoa of Young Men Living in the "Valley of Sacco River" (Latium, Italy): A Preliminary Study.

The Valley of Sacco River (VSR) (Latium, Italy) is an area with large-scale industrial chemical production that has led over time to significant contamination of soil and groundwater with various industrial pollutants, such as organic pesticides, dioxins, organic solvents, heavy metals, and particularly, volatile organic compounds (VOCs). In the present study, we investigated the potential impact of VOCs on the spermatozoa of healthy young males living in the VSR, given the prevalent presence of several VOCs in the semen of these individuals. To accomplish this, spermiograms were conducted followed by molecular analyses to assess the content of sperm nuclear basic proteins (SNBPs) in addition to the protamine-histone ratio and DNA binding of these proteins. We found drastic alterations in the spermatozoa of these young males living in the VSR. Alterations were seen in sperm morphology, sperm motility, sperm count, and protamine/histone ratios, and included significant reductions in SNBP-DNA binding capacity. Our results provide preliminary indications of a possible correlation between the observed alterations and the presence of specific VOCs.