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This review provides details on the role of centrin in human spermatozoa and in various forms of male infertility. Centrin is a calcium (Ca2+)-binding phosphoprotein that is located in the centrioles – which are typical structures of the sperm connecting piece and play a key role in centrosome dynamics during sperm morphogenesis – as well as in zygotes and early embryos during spindle assembly. In humans, three different centrin genes encoding three isoforms have been discovered. Centrin 1, the only one expressed in spermatozoa, seems to be lost inside the oocyte after fertilization. The sperm connecting piece is characterized by the presence of numerous proteins including centrin, that deserves particular attention because, in humans, it is enriched during maturation of the centrioles. In normal sperm, centrin 1 is visible as two distinct spots in the head–tail junction; however, in some defective spermatozoa, centrin 1 distribution is altered. Centrin has been studied in humans and animal models. Its mutations may lead to several structural alterations, such as serious defects in the connective piece and, subsequently, fertilization failure or incomplete embryonic development. However, the effects of these abnormalities on male fertility have not been fully studied. Because the presence and the function of centrin in the sperm connecting piece appears important for reproductive success, additional studies are needed to bring medical benefits in resolving some cases of idiopathic infertility. [ABSTRACT FROM AUTHOR]

Liposomes have been successfully used as drug-delivery vehicles, but there are no clinical studies on improved fertility and the few reported experimental studies have been performed in animal models far from humans. The aim of this paper was to study the effects of treatment with cationic, anionic and zwitterionic liposomes on our superior mammalian model of porcine prepubertal Sertoli cells (SCs) to find a carrier of in vitro test drugs for SCs. Porcine pre-pubertal SCs cultures were incubated with different liposomes. Viability, apoptosis/necrosis status (Annexin-V/Propidium iodide assay), immunolocalisation of β-actin, vimentin, the phosphorylated form of AMP-activated protein Kinase (AMPK)α and cell ultrastructure (Transmission Electron Microscopy, TEM) were analysed. Zwitterionic liposomes did not determine changes in the cell cytoplasm. The incubation with anionic and cationic liposomes modified the distribution of actin and vimentin filaments and increased the levels of the phosphorylated form of AMPKα. The Annexin/Propidium Iodide assay suggested an increase in apoptosis. TEM analysis highlighted a cytoplasmic vacuolisation. In conclusion, these preliminary data indicated that zwitterionic liposomes were the best carrier to use in an in vitro study of SCs to understand the effects of molecules or drugs that could have a clinical application in the treatment of certain forms of male infertility. [ABSTRACT FROM AUTHOR]

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]

This brief report concerns the role of human sperm morphology assessment in different fields of male infertility: basic research, genetics, assisted reproduction technologies, oxidative stress. One of the best methods in studying sperm morphology is transmission electron microscopy (TEM) that enables defining the concept of sperm pathology and classifying alterations in non-systematic and systematic. Non-systematic sperm defects affect head and tail in variable ratio, whereas the rare systematic defects are characterized by a particular anomaly that marks most sperm of an ejaculate. TEM analysis and fluorescence in situ hybridization represent outstanding methods in the study of spermmorphology and cytogenetic in patients with altered karyotype characterizing their semen quality before intracytoplasmic sperm injection. In recent years, the genetic investigations on systematic sperm defects, made extraordinary progress identifying candidate genes whose mutations induce morphological sperm anomalies. The question if sperm morphology has an impact on assisted fertilization outcome is debated. Nowadays, oxidative stress represents one of themost important causes of altered spermmorphology and function and can be analyzed from two points of view: 1) spermatozoa with cytoplasmic residue produce reactive oxygen species, 2) the pathologies with inflammatory/oxidative stress background cause morphological alterations. Finally, sperm morphology is also considered an important endpoint in in vitro experiments where toxic substances, drugs, antioxidants are tested. We think that the field of spermmorphology is far frombeing exhausted and needs other research. This parameter can be still considered a valuable indicator of sperm dysfunction both in basic and clinical research. [ABSTRACT FROM AUTHOR]

In this review, the role of fatty acids (FA) in human pathological conditions, infertility in particular, was considered. FA and FA-derived metabolites modulate cell membrane composition, membrane lipid microdomains and cell signaling. Moreover, such molecules are involved in cell death, immunological responses and inflammatory processes. Human health and several pathological conditions are specifically associated with both dietary and cell membrane lipid profiles. The role of FA metabolism in human sperm and spermatogenesis has recently been investigated. Cumulative findings indicate F2 isoprostanes (oxygenated products from arachidonic acid metabolism) and resolvins (lipid mediators of resolution of inflammation) as promising biomarkers for the evaluation of semen and follicular fluid quality. Advanced knowledge in this field could lead to new scenarios in the treatment of infertility. [ABSTRACT FROM AUTHOR]

Purpose: Omentin-1/intelectin (ITLN)1 is an adipocytokine with both anti-inflammatory and anti-oxidative stress properties, and little is known about its role in male reproduction. This study was aimed at exploring the relationships among omentin-1/ITLN1, semen parameters and F2-isoprostanes (F2-IsoPs), a maker of oxidative stress, in groups of patients affected by different pathologies. In addition, omentin-1/ITLN1 immunolocalization was assessed in ejaculated spermatozoa and in tissues of male reproductive system. Patients and Methods: Semen samples of infertile patients with varicocele (n = 27), genitourinary infections (n = 17), idiopathic infertility (n = 15) and fertile men (n = 21) were analyzed following WHO guidelines, and seminal plasma were used to determine omentin-1/ITLN1 by ELISA and F2-IsoP levels by gas chromatography/negative-ion chemical ionization tandem mass spectrometry. Omentin-1/ITLN1 was localized in human sperm and in the tissue of male reproductive system. Results: Considering all participants, F2-IsoP and omentin-1/ITLN1 levels were positively correlated (p = 0.000), and both these indices were negatively correlated with sperm parameters. Infertile patients showed lower sperm parameters than fertile ones; varicocele and infection groups had significantly increased levels of F2-IsoPs (both p = 0.000) and omentin-1/ITLN1 (p = 0.000 and p = 0.001, respectively). Omentin-1/ITLN1 signal was located as a spot in the connecting piece (in 43.5% of cases midpiece was also labeled) of sperm from fertile men and in cytoplasmic residue and in the entire tail in sperm of patients with varicocele and genitourinary infections. A focal omentin-1/ITLN1 immunolabelling was evident in the basal area of epididymal tubule, and a diffuse signal was present in the seminal vesicle epithelium. Conclusion: Semen omentin-1/ITLN1 originates from seminal vesicles, its levels increase in inflammatory conditions and are negatively correlated with sperm parameters. For this reason, a sort of protective role of omentin-1/ITLN1 can be postulated, as this adipokine shows anti-inflammatory properties also in many other biological systems. Graphical [ABSTRACT FROM AUTHOR]

In cancer survivors, chemotherapy-associated adverse neurological effects are described as side effects in non-targeted tissue. We investigated the role of redox-imbalance in neuronal damage by a relative low dose of Docetaxel (DTX). The neuroblastoma cells (SH-SY5Y cells) were exposed to DTX at a dose of 1.25 nM for 6 h. Antioxidant defenses (i.e. ascorbic acid, glutathione, and catalase) and lipid oxidation products (i.e. F2-isoprostanes) were evaluated. To investigate cell ultrastructure and tubulin localisation, transmission electron microscopy (TEM) and immunofluorescence techniques were applied. In the SH-SY5Y cells, DTX induced a significant reduction of total glutathione (P < 0.001) and ascorbic acid (P < 0.05), and an increase in both total F2-Isoprostanes (P < 0.05) and catalase activity (P < 0.05), as compared to untreated cells. Additionally, TEM showed a significant increase in cells with apoptotic characteristics. Immunolocalisation of tubulin showed a compromised cytoskeletal organisation. The investigated sublethal dose of DTX, to which non-targeted cells may be exposed throughout the duration of chemotherapy treatment, induces a redox imbalance resulting in a specific modulation of the antioxidant response. This study provides new insights into DTX-induced cellular mechanisms useful for evaluating whether the concomitant use of antioxidants associated with chemotherapy mitigates chemotherapy side effects in cancer survivors. [ABSTRACT FROM AUTHOR]

Objectives. Fatty acids (FA) modulate oxidative stress, reactive oxygen species (ROS) production, and inflammatory processes in spermatogenesis. Methods. The amount of 17 different FAs and the level of F2-isoprostanes (F2-IsoPs) and cytoplasmic phospholipase A2 (cPLA2) were compared and correlated to sperm characteristics; these last ones were evaluated by light and electronic microscopy in varicocele and idiopathic infertile patients. Results. Total n-3 polyunsaturated acids (PUFAs) and docosahexaenoic acid (DHA), one of the n-3 PUFAs, were significantly reduced in idiopathic infertile men compared to controls (P < 0.05). In the whole studied population, oleic acid and total monounsaturated acids (MUFAs) correlated negatively with sperm concentration, progressive motility, normal morphology, vitality, and fertility index and positively with sperm necrosis. Eicosapentaenoic acid (EPA) amount was positively correlated with the percentage of sperm necrosis and cPLA2 level and negatively with sperm concentration. Sperm vitality was negatively correlated with the saturated fatty acids (SFAs). In infertile groups, cPLA2 was negatively correlated with DHA and n-3 PUFAs (both P < 0.05) and positively with EPA (P < 0.05). In the varicocele group, sperm vitality was negatively correlated with palmitoleic acid and total n-6 PUFAs (P < 0.05); sperm apoptosis was positively correlated with the total SFA percentage (P < 0.05). Conclusions. FA composition in sperm membrane and the metabolism of sperm FAs are interrelated parameters, both relevant in sperm maturation processes and fertility. [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]

The aim of this study was to clarify the role of the protein kinase suppressor of Ras1 (KSR1) in spermatogenesis. Spermatogenesis in ksr1 −/− mice was studied in testicular tissue and epididymal spermatozoa by light and transmission electron microscopy and by immunofluorescence using antibodies to ghrelin and 3β-hydroxysteroid dehydrogenase (3β-HSD). Blood testosterone levels were also assessed. ksr1 −/− mice showed reduced epididymal sperm concentration and motility as compared with wild - type (wt) mice. Testis tissue from ksr1 −/− mice revealed a prevalent spermatogenetic arrest at the spermatocyte stage; the interstitial tissue was hypertrophic and the cytoplasm of the Leydig cells was full of lipid droplets. Ghrelin signal was present in the seminiferous tubules and, particularly, in the interstitial tissue of wt mice; however, in ksr1 −/− mice ghrelin expression was very weak in both the interstitial tissue and tubules. On the contrary, the signal of 3β-HSD was weak in the interstitial tissue of wt and strong in ksr1 −/− mice. Testosterone levels were significantly increased in the blood of ksr1 −/− mice (P < 0.05) as compared with wt. The results obtained reveal the importance of the KSR scaffold proteins in the spermatogenetic process. The study of the molecular mechanisms associated with spermatogenetic defects in a mouse model is essential to understand the factors involved in human spermatogenesis. Kinase suppressor of Ras1 (ksr1)−/− mice show spermatocyte stage arrest in most tubules and altered Leydig cells. The ksr1 gene encodes for a scaffolding protein (KSR1) involved in the signal transduction pathway. The spermatogenetic arrest was concomitant with low expression of ghrelin, a protein involved in reproduction, high expression of 3β-HSD, which is involved in steroid synthesis, and high blood testosterone level. KSR1, which is involved in the mouse spermatogenetic process, could play a role in human male fertility. [ABSTRACT FROM AUTHOR]

F2-isoprostanes (F2-IsoPs), stereoisomers of prostaglandin F2α generated by the free radical-induced oxidation of arachidonic acid, have been associated with different male infertility conditions. This study aimed to evaluate the role of seminal isoprostane levels and sperm characteristics in the reproductive outcome and embryo quality of 49 infertile couples. Semen analysis was performed following WHO guidelines. Sperm chromatin maturity was detected using an aniline blue (AB) assay, and DNA integrity was assessed using the acridine orange (AO) test. Seminal F2-IsoP levels were quantified by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI–MS/MS) analysis. Correlations among variables and their impact on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcome were investigated. F2-IsoP levels are positively correlated with double-stranded DNA sperm (p < 0.001) and negatively correlated with mature sperm chromatin (p < 0.001). Patients with positive outcomes had an increased percentage of sperm with double-stranded DNA, as did patients producing high-quality embryo, who showed higher F2-IsoP levels compared to those detected in the low-quality embryo group. An intriguing relationship between a mild increase in F2-IsoP levels, DNA integrity, and embryo quality seems to indicate that the non-enzymatic oxidation of arachidonic acid can be also a marker of metabolic activity in human semen. [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]

The study evaluated the chlorogenic acid (CGA) antioxidant potential on oxidative stress (OS) induced in vitro in human spermatozoa and during cryopreservation procedure. Swim-up selected spermatozoa were treated with 100 µM CGA, 100 µM H2O2 to induce lipid peroxidation (LPO), and with both compounds and the effects on mitochondrial membrane potential (MMP) by JC-1, DNA integrity by acridine orange (AO), and sperm ultrastructure by transmission electron microscopy (TEM), were evaluated. CGA antioxidant activity was assessed by measuring malondialdehyde (MDA) and F2-isoprostanes (F2-IsoPs) in the media. The CGA protective activity and the immunolocalization of Phospho-AMPKα (Thr172) were explored in frozen-thawed sperm. CGA was not toxic for sperm motility, DNA integrity and MMP. The increase in MDA (p < 0.05) and F2-IsoPs (p < 0.001), DNA damage (p < 0.01) and low MMP (p < 0.01) levels after H2O2 treatment were reduced in presence of CGA as well as the percentage of broken plasma membranes (p < 0.01) and altered acrosomes (p < 0.01) detected by TEM. Treated frozen-thawed spermatozoa showed increased sperm motility (p < 0.01), DNA integrity (p < 0.01), MMP (p < 0.01), reduced MDA (p < 0.01) and increased sperm percentage with Phospho-AMPKα labelling in the head (p < 0.001). CGA can be used to supplement culture media during semen handling and cryopreservation where OS is exacerbated. [ABSTRACT FROM AUTHOR]

Polyunsaturated fatty acid (PUFA) metabolism and tissue distribution is modulated by the oxidation of these molecules. This research aimed to investigate the implication of dietary n-3 PUFA supplementation (precursor and long-chain PUFA) on the PUFA profile and oxidative status of the liver, testis, and brain of adult rabbit bucks. Twenty New Zealand White rabbit bucks were divided into four experimental groups (n = 5 per group) and were fed different diets for 110 days: control (CNT), standard diet containing 50 mg/kg alpha-tocopheryl acetate (vitamin E); CNT+, standard diet + 200 mg/kg vitamin E; FLAX, standard diet + 10% flaxseed + 200 mg/kg vitamin E; or FISH, standard diet + 3.5% fish oil + 200 mg/kg vitamin E. Antioxidants (enzymatic and non-enzymatic), oxidative status (malondialdehyde and isoprostanoids), and n-3 and n-6 PUFAs of tissues were analysed. A chain mechanism of oxidant/antioxidant molecules, which largely depended on the particular PUFA composition, was delineated in the different organs. The liver showed an oxidant/antioxidant profile and lipid pathways widely modulated by PUFA and vitamin E administration; on the other hand, the testis' oxidative profile rather than its lipid profile seemed to be particularly affected, an outcome opposite to that of the brain (modulation operated by dietary PUFA). [ABSTRACT FROM AUTHOR]

Specialized pro-resolving lipid mediators regulate the resolution of acute inflammation. They are formed by enzymatic oxygenation of polyunsaturated fatty acids and are divided into families including lipoxins, resolvins, protectins, and maresins. Resolvin D1 (RvD1), produced by docosahexaenoic acid, exerts anti-inflammatory and pro-resolving activities. This research aimed to investigate the implication of seminal RvD1 in human infertility. Infertile patients (n° 67) were grouped based on pathological reproductive conditions as idiopathic infertility, varicocele, and leukocytospermia; the fourth group was composed of fertile men (n° 18). Sperm characteristics were evaluated by light microscopy (WHO guidelines) and by transmission electron microscopy (TEM). The seminal levels of RvD1 and F 2 -isoprostane (F 2 -IsoPs) were dosed. In twenty men (6 fertile men, 8 with varicocele, 6 with leukocytospermia) seminal phospholipase A 2 , iron, cholesterol, transferrin, estradiol, ferritin, testosterone, and sperm membrane fatty acids were detected. The results indicated that: (i) RvD1 amount was positively correlated with F 2 -IsoPs and reduced sperm quality; (ii) RvD1 levels were significantly higher in patients with leukocytospermia, varicocele, and idiopathic infertility compared to fertile men; (iii) RvD1 increased along with other markers of oxidative stress and inflammation as fatty acids content and clinical biomarkers. This study suggests a panel of inflammatory markers and lipid mediators for a diagnosis of inflammatory status and a subsequent appropriate therapeutic approach.

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.)