Prostasomes are exosomes such as extracellular vesicles, produced in the prostatic epithelium and released into the seminal plasma, that play an important role enhancing male fertility. Although some studies have demonstrated that prostasomes have a rich proteomic content, it is still unclear if that proteomic content varies depending on the male fertility status. Prostasomes from 12 normozoospermic and 14 non-normozoospermic seminal samples were isolated by differential ultracentrifugation. Protein content was studied by quantitative mass spectrometry and compared between both cohorts. We identified 1282 proteins with 745 of them (57.8%) being present in all seven prostasome pools. Forty-seven of those commonly present proteins showed differential expression levels in both cohorts. Specifically, prostasomes from non-normozoospermic samples showed a pattern of protein underexpression for a group of proteins including several proteins from the spermatozoa's energy production pathways as well as some proteins directly implicated in sperm activity. Variations in prostasomal protein content levels may have a relevant correlation with male fertility and thus could be of great utility as a biomarker of fertility status.
The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37C showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions.
The DNA repair capacity in the mature spermatozoa is highly compromised due to the base-excision repair (BER) route being truncated. In the mature spermatozoa, only the first enzyme of the route (OGG1) is present. Consequently, reduced activity of the enzymes of the BER route both during spermatogenesis and in the mature spermatozoa may be detrimental for fertility. The objective of our study was to investigate the correlation between two representative SNPs of those enzymes, SNPs OGG1 Ser326Cys (rs1052133) and XRCC1 Arg399Gln (rs25487) and male infertility. A total of 313 seminal samples from infertile patients and 80 from donors with proven fertility were included in the study. All samples were subjected to a regular sperm analysis and genotyped using the PCR-RFLP system. We found significant differences in the genotype frequencies between patients and donors for the XRCC1 Arg399Gln polymorphism (χ2(2) = 8.7, p = 0.013), with the Gln allele showing a protective role and for the OGG1 Ser326Cys polymorphism between normozoospermic and non-normozoospermic patients (χ2(2) = 12.67, p = 0.002) with the Cys allele showing a detrimental effect over concentration. In conclusion, our study shows that polymorphisms in the genes coding for the DNA damage repair enzymes may be associated with poor sperm parameters and male infertility.
IntroductionExtracellular vesicles (EVs) have been recognized as key players in numerous physiological functions. These vesicles alter their compositions attuned to the health and disease states of the organism. In men, significant changes in the proteomic composition(s) of seminal plasma EVs (sEVs) have already been found to be related to infertility.MethodsMethods: In this study, we analyze the posttranslational configuration of sEV proteomes from normozoospermic (NZ) men and non-normozoospermic (non-NZ) men diagnosed with teratozoospermia and/or asthenozoospermia by unbiased, discovery-driven proteomics and advanced bioinformatics, specifically focusing on citrulline (Cit) and homocitrulline (hCit) posttranscriptional residues, both considered product of ureido protein modifications. Results and discussionSignificant increase in the proteome-wide cumulative presence of hCit together with downregulation of Cit in specific proteins related to decisive molecular functions have been encountered in sEVs of non-NZ subjects. These findings identify novel culprits with a higher chance of affecting fundamental aspects of sperm functional quality and define potential specific diagnostic and prognostic non-invasive markers for male infertility.
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