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Swim-up selected human sperm were incubated with 7 ng F4-neuroprostanes (F4-NeuroPs) for 2 and 4 h. Sperm motility and membrane mitochondrial potential (MMP) were evaluated. The percentage of reacted acrosome was assessed by pisum sativum agglutinin (PSA). Chromatin integrity was detected using the acridine orange (AO) assay and localization of the ryanodine receptor was performed by immunofluorescence analysis. Sperm progressive motility (p = 0.02) and the percentage of sperm showing a strong MMP signal (p = 0.012) significantly increased after 2 h F4-NeuroP incubation compared to control samples. The AO assay did not show differences in the percentage of sperm with dsDNA between treated or control samples. Meanwhile, a significantly higher number of sperm with reacted acrosomes was highlighted by PSA localization after 4 h F4-NeuroP incubation. Finally, using an anti-ryanodine antibody, the immunofluorescence signal was differentially distributed at 2 and 4 h: a strong signal was evident in the midpiece and postacrosomal sheath (70% of sperm) at 2 h, whereas a dotted one appeared at 4 h (53% of sperm). A defined concentration of F4-NeuroPs in seminal fluid may induce sperm capacitation via channel ions present in sperm cells, representing an aid during in vitro sperm preparation that may increase the positive outcome of assisted fertilization. [ABSTRACT FROM AUTHOR]

Phospholipase A2 (PLA2) is involved in eicosanoid release, and F2‐isoprostanes (F2‐IsoPs), as free radical‐generated eicosanoids released by PLA2, are indicators of oxidative stress in different human conditions. This study investigated the interplay between cytosolic PLA2 (cPLA2), F2‐IsoPs and sperm features in male infertility, when the involvement of oxidative stress has been reported. Semen evaluation was performed following WHO guidelines, sperm ultrastructure was detected by transmission electron microscopy indicating a fertility index, and the percentages of sperm immaturity, apoptosis and necrosis. In sperm cells and seminal plasma, cPLA2 levels were determined by immunological method, whereas F2‐IsoPs by mass spectrometry. Sperm concentration, morphology, vitality and fertility index values were significantly lower in infertile groups compared with fertile men. An increase in sperm apoptosis and necrosis (p <.01), apoptosis (p <.01) and immaturity (p <.001) was detected in leucocytospermia, idiopathic infertility and varicocele, respectively. Seminal cPLA2 showed the highest value in varicocele group (p <.05), whereas seminal F2‐IsoPs increased in varicocele (p <.001) and leucocytospermia (p <.05) groups. In the whole population, F2‐IsoP and cPLA2 levels were positively correlated (p <.05). On the contrary, F2‐IsoPs and cPLA2 were not significantly different when investigated in sperm cells. Our data indicate that fatty acid oxidation/metabolism plays a role in different male reproductive pathological conditions. [ABSTRACT FROM AUTHOR]

F4-neuroprostanes (F4-NeuroPs), derived from the oxidative metabolization of docosahexaenoic acid (DHA), are considered biomarkers of oxidative stress in neurodegenerative diseases. Neurons and spermatozoa display a high DHA content. NeuroPs might possess biological activities. The aim of this in vitro study was to investigate the biological effects of chemically synthetized 4-F4t-NeuroP and 10-F4t-NeuroP in human sperm. Total progressive sperm motility (p < 0.05) and linearity (p = 0.016), evaluated by a computer-assisted sperm analyzer, were significantly increased in samples incubated with 7 ng F4-NeuroPs compared to non-supplemented controls. Sperm capacitation was tested in rabbit and swim-up-selected human sperm by chlortetracycline fluorescence assay. A higher percentage of capacitated sperm (p < 0.01) was observed in samples incubated in F4-NeuroPs than in the controls. However, the percentage of capacitated sperm was not different in F4-NeuroPs and calcium ionophore treatments at 2 h incubation. The phosphorylated form of AMPKα was detected by immunofluorescence analysis; after 2 h F4-NeuroP incubation, a dotted signal appeared in the entire sperm tail, and in controls, sperm were labeled in the mid-piece. A defined level of seminal F4-NeuroPs (7 ng) showed a biological activity in sperm function; its addition in sperm suspensions stimulated capacitation, increasing the number of sperm able to fertilize. [ABSTRACT FROM AUTHOR]

In the last two decades, the human sperm count linearly decreased in Western countries. Health problems, lifestyle, pollutants, and dietary behaviours are considered as the main risk factors, and the unbalance of dietary n -6/ n -3 fatty acids is one of the most relevant. The aim of the present research is to study the effect of different dietary sources of n -3 polyunsaturated fatty acids (PUFA) on reproductive traits using rabbit buck as the animal model. Fifteen rabbit bucks were assigned to three experimental groups: the control group, the FLAX group fed 10% extruded flaxseed, and the FISH group fed 3.5% fish oil for 110 days (50-day adaptation and 60-day experimental periods). Semen samples were collected weekly, whereas blood was collected every two weeks for the analytical determination of semen traits, oxidative status, fatty acid profiles, isoprostanes, neuroprostanes, and the immunocytochemistry of docosahexaenoic acid (DHA) and eicosapentaenoic (EPA) acid. At the end of the trial, the rabbits were killed and the testes were removed and stored for the analysis of fatty acid profile and immunocytochemistry. Results showed that dietary administration of n -3 PUFA improved the track speed of the sperm and increased the n -3 long-chain PUFA mainly confined in the sperm tail. Seminal plasma increased the thiobarbituric reactive substances (TBARs) by three times in the groups fed supplemental n -3, whereas the F 2 -isoprotanes (F 2 -IsoPs) and F 4 -neuroprostanes (F 4 -NeuroPs) were lower and higher, respectively, in both supplemented groups than in the control. The testes and sperm showed a higher DHA and EPA distribution in rabbits from the n -3 supplemented groups compared with the control. In conclusion, supplemental dietary n -3 PUFA improved sperm motion traits and resulted in an enrichment of membrane fatty acid in the sperm and testes of the rabbits. However, such an increased amount of PUFA negatively affected the sperm oxidative status, which was mainly correlated with the generation of F 4 -NeuroPs with respect to F 2 -IsoPs. Accordingly, the latter cannot be considered a good marker of oxidation when diets rich in n -3 PUFA are provided., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Cesare Castellini et al.)