Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers

Abstract

ObjectiveTo evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. Patients and MethodsSperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatographytandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in. ResultsSmokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokinemediated signalling pathway and regulation of acute inflammatory response in the study group (smokers). ConclusionIn conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality, which is characterized by decreased acrosome integrity and mitochondrial activity, as well as by increased nuclear DNA fragmentation.

Resveratrol attenuates reproductive alterations in type 1 diabetes‐induced rats

“…This methodology is based on the oxidation of 3,3′‐diaminobenzidine (DAB), by the enzyme cytochrome c‐oxidase, through a chain reaction in which the reagent is polymerized and deposited in the midpiece of the spermatozoa (Antoniassi et al . ; Miranda‐Spooner et al . ).…”

The most common vices of men can damage fertility and the health of the next generation

“…Smokers are at increased risk of sperm defects such as partially or fully inactive mitochondria and non-intact acrosomes, in addition to changes to the proteome of their seminal plasma (Antoniassi et al 2016). These changes to DNA and seminal plasma are hypothesised to further contribute to the increased childhood cancer risk seen in children born to male smokers, beyond the contribution of paternal smoking to the increased mutation load in both the father's germline and transmitted to his children (Linschooten et al 2013).…”

Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking

“…Sperm nuclear DNA fragmentation was evaluated by an alkaline Comet assay (Antoniassi et al, 2016). Briefly, slides were previously coated with 1% normal melting point agarose diluted in TBE (0.089 M Tris, 0.089 M borate and 0.002 M EDTA) until completely dry.…”

“…Mitochondrial activity was performed by staining with 3,3′‐diaminobenzidine (DAB; Antoniassi et al, 2016; Intasqui et al, 2015). This technique is based on the dye oxidation by the cytochrome C complex and the following deposition in the sperm midpiece/mitochondria (Hrudka, 1987).…”

“…Smoking alone has been controversially associated with a decrease in semen quality (Böhlandt et al, 2012; Fariello et al, 2012; Oteiza, Olin, Fraga, & Keen, 1996; Potts, Newbury, Smith, Notarianni, & Jefferies, 1999; Saleh, Agarwal, Sharma, Nelson, & Thomas, 2002; Sofikitis et al, 1995), as well as sperm functional analysis (Antoniassi et al, 2016; Fariello et al, 2012; Potts et al, 1999; Saleh et al, 2002; Sofikitis et al, 1995). However, information about the precise protein environment from these patients associated with varicocele is still lacking.…”