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[EN] Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pH(i)) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4-7.6. The natural pH(i) range of sperm samples in the quiescent stage was 7.4-8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pH(e)), as well as between the pH(i) and the pH of the diluent. The change post-activation in seawater (ASW) depended on the initial pH(e) of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pH(e) 8.0; an acidification when pH(e) > 8, and no pH(i) variation when pH(e) was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pH(i) changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation. (C) 2020 Elsevier Inc. All rights reserved. ; Funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS). Victor Gallego has a postdoc grant from the MICIU (Juan de la Cierva-Incorporacion; IJCI-2017-34200 ; Pérez Igualada, LM.; Gallego Albiach, V.; Asturiano, JF. (2020). Intracellular pH regulation and sperm motility in the European eel. Theriogenology. 145:48-58. ...

The relationship between dioxin-like polychlorinated biphenyl (DL˗PCB) levels in serum and semen parameters were investigated. Our case-control included two groups of patients. Total concentrations of PCBs were significantly higher in the low semen quality (n = 24) than in the normal semen quality (n = 26) group. A significant negative correlation was found between PCB 126 and viability in men with low semen quality, while PCBs 77 and 81 were positively correlated with morphology, and PCB 118, mono-ortho and total DL-PCBs were positively correlated with volume. In the normal semen quality group, PCB 189 and 118 were negatively correlated with sperm motility and volume, respectively. In addition, positive significant correlations were found between PCB 77, 23 and total non-ortho PCBs with regard to morphology. Our findings suggest that sperm motility, viability, volume and morphology are parameters sensitive to alteration by exposure to DL-PCBs, although PCB effects on spermatogenesis were not of clinical significance. ; This study was supported by Cátedra Human Fertility from the University of Alicante, CTQ2013-41006-R grant from the Ministerio Economía y Competitividad, and PROMETEOII/2014/007 grant from the Valencian Community Government (Spain).

14 Pág. ; The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P P P. ; This research was funded by the INIA project RTA2017-00034-00-00. Part of this research received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 677353 IMAGE. ; Peer reviewed

[EN] During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka¿s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation. ; This publication was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 642893 (IMPRESS), the COST Office (COST Action FA 1205, AQUAGAMETE), and the Research Centre of Excellence -1476-4/2016/FEKUT ; Herranz-Jusdado, JG.; Kása, É.; Kollár, T.; Gallego Albiach, V.; Peñaranda, D.; Rozenfeld, C.; Pérez Igualada, LM. (2018). Handling and treatment of male European eels (Anguilla anguilla) for hormonal maturation and sperm cryopreservation. Journal of Visualized Experiments. 131(e56835):1-6. https://doi.org/10.3791/56835 ; S ; 1 ; 6 ; 131 ; e56835

AbstractHuman semen contains several trace elements such as calcium (Ca), copper (Cu), manganese (Mn), magnesium (Mg), zinc (Zn) and selenium (Se) which are necessary for reproductive health, normal spermatogenesis, sperm maturation, motility and capacitation, as well as normal sperm function. In this review, the potential role of these trace elements in male reproductive health, normal function of spermatozoa and fertility potency were considered. We selected and reviewed articles that considered crucial roles of trace elements in human sperm function and fertility. Ca is essential for sperm motility and its hyperactivation, sperm capacitation and acrosome reaction, as well as sperm chemotaxis. Sodium (Na) and potassium (K) are involved in sperm motility and capacitation. Mg is necessary for normal ejaculation, spermatogenesis and sperm motility. Zn is one of the most significant nutrients in human semen. Seminal deficiency of Zn can be associated with delayed testicular development, impaired spermatogenesis, deficiency of sex hormones, oxidative stress and inflammation, and apoptosis. Se is another significant element which has antioxidative properties and is essential for spermatogenesis and the maintenance of male fertility. Mn is a potent stimulator for sperm motility; however, increased level of seminal plasma Se can be toxic for sperm. Like Se, Cu has antioxidative properties and has a positive effect on sperm parameters. Decreased level of these trace elements can negatively affect human reproductive health, semen quality, sperm normal function and as the result, fertility potency in men. Measurement of these trace elements in men with idiopathic infertility is necessary.

BACKGROUND: Sperm maturation takes place through contact between sperm and proteins produced in the epididymal lumen. CD52 had been characterised in the sperm; however, the expression and its regulation in the epididymis are mostly unknown. AIM: This study aimed to analyse the expression and regulation of CD52 in the mouse epididymis. SETTING AND DESIGN: Experimental design was used in this study. MATERIALS AND METHODS: Epididymis tissues from mice strain Deutch Democratic Yokohama were used as sources of total RNA. Bioinformatic tool was used to predict signal peptides. Quantitative real-time reverse transcription–polymerase chain reaction was used to analyse tissue distribution, androgen, testicular factors dependency and postnatal development. STATISTICAL ANALYSIS: One-way analysis of variance was used to analyse differences between treatment and control untreated group. P < 0.05 was determined as a significant difference. RESULTS: CD52 amino acid sequence contains a signal peptide, indicating it is a secretory protein. CD52 exhibited region-specific expression in the epididymis, with the highest level being in the cauda. CD52 expression was regulated by androgen indicated by a significant downregulation at day 1 and day 3 following a castration (P < 0.05). Dependency on androgen was confirmed by injection of exogenous testosterone which prevented downregulation by 50%. Moreover, lumicrine factors also influenced CD52 expression indicated by ligation of efferent duct which also reduced expression at day 1 to day 5 following the ligation (P < 0.05). CD52 expression was developmentally regulated. This was shown by increase in the level of expression starting at day 15 postnatally. CONCLUSION: CD52 shows characteristics of genes involved in sperm maturation in the epididymis.

AbstractDNA damage in spermatozoa is a major cause of male infertility. It is also associated with adverse reproductive outcomes (including reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage). The damage to sperm DNA occurs during the production and maturation of spermatozoa, as well as during their transit through the male reproductive tract. DNA damage repair typically occurs during spermatogenesis, oocytes after fertilization, and early embryonic development stages. The known mechanisms of sperm DNA repair mainly include nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and double-strand break repair (DSBR). The most severe type of sperm DNA damage is double-strand break, and it will be repaired by DSBR, including homologous recombination (HR), classical non-homologous end joining (cNHEJ), alternative end joining (aEJ), and single-strand annealing (SSA). However, the precise mechanisms of DNA repair in spermatozoa remain incompletely understood. DNA repair-associated proteins are of great value in the repair of sperm DNA. Several repair-related proteins have been identified as playing critical roles in condensing chromatin, regulating transcription, repairing DNA damage, and regulating the cell cycle. It is noteworthy that XRCC4-like factor (XLF) and paralog of XRCC4 and XLF (PAXX) -mediated dimerization promote the processing of populated ends for cNHEJ repair, which suggests that XLF and PAXX have potential value in the mechanism of sperm DNA repair. This review summarizes the classic and potential repair mechanisms of sperm DNA damage, aiming to provide a perspective for further research on DNA damage repair mechanisms.

It is well-established that testicular spermatozoa are immature and acquire motility and fertilization capabilities during transit throughout the epididymis. The epididymis is a duct-like organ that connects the testis to the vas deferens and is comprised of four anatomical regions: the initial segment, caput, corpus, and cauda. Sperm maturation occurs during epididymal transit by the interaction of sperm cells with the unique luminal environment of each epididymal region. In this review we discuss the epididymis as an essential reproductive organ responsible for sperm concentration, maturation (including sperm motility acquisition and fertilizing ability), protection and storage. Importantly, we also discuss specific characteristics and roles of epididymal-derived exosomes (epididymosomes) in establishing sperm competency within the intricate process of reproduction. This review suggests that an increasing body of evidence is working to develop a complete picture of the role of the epididymis in male reproduction, offspring health, and disease susceptibility ; The authors acknowledge the support from the Ministry of Science and Innovation, Spain (Grants: RYC–2014–15581 and AGL2017–88329–R) and the Regional Government of Catalonia, Spain (Grant: 2019–SGR–1229)

European Union (Marie Curie International Incoming Fellowship) ; European Research Council Advanced Grant ; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ; Finnish Cultural Foundation ; Academy of Finland ; Tampere University Medical Research Fund ; Sigrid Juselius Foundation ; European Union (Marie Curie International Incoming Fellowship): GA328988 ; European Research Council Advanced Grant: 232,738 ; Processo FAPESP: 2014/02253-6 ; Academy of Finland: 256,615 ; Academy of Finland: 272,376 ; Background: Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question. Methods: Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the a-tubulin promoter, and wild-type males of the same genetic background. Results: In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation. Conclusions: AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.

Aldose reductase B1 (AKR1B1) has been reported to participate in the modulation of male and female reproductive physiology in several mammalian species. In spite of this, whether or not AKR1B1 could be related to sperm quality, functionality and fertilizing ability is yet to be elucidated. The present study, therefore, aimed to investigate: i) the presence of AKR1B1 in epididymal and ejaculated sperm; ii) the relationship between the AKR1B1 present in sperm and the physiology of the male gamete; iii) the liaison between the relative content of AKR1B1 in sperm and their ability to withstand preservation for 72 h; and iv) the potential link between sperm AKR1B1 and in vitro fertility outcomes. Immunoblotting revealed that AKR1B1 is present in both epididymal and ejaculated sperm with a similar relative content. Moreover, the relative levels of AKR1B1 in sperm (36 kDa band) were found to be negatively related to several kinematic parameters and intracellular calcium levels, and positively to the percentage of sperm with distal cytoplasmic droplets after storage. Finally, AKR1B1 amounts in sperm (36 kDa band) were negatively associated to fertilization rate at two days post-fertilization and embryo development at six days post-fertilization. The results of the present work suggest that AKR1B1 in sperm is probably acquired during maturation rather than at ejaculation and could play a role in that process. Moreover, AKR1B1 seems to be related to the sperm resilience to preservation and to their fertilizing capacity, as lower levels of the 36 kDa band (putative inactive form of this protein) result in better reproductive outcomes ; The present study was funded by the Ministry of Science and Innovation, Spain (AGL2017-88329-R and FJCI-2017-31689), the Regional Government of Catalonia, Spain (2017-SGR-1229 and 2020-FI-B-00412) and the European Commission (H2020 Research and Innovation program under the Marie SkłodowskaCurie grant agreement No 801342; Tecniospring INDUSTRY, TECSPR-19-1-0003)

<p>Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the <em>in vitro </em>maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P&gt;0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P&lt;0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P&lt;0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P&lt;0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P&lt;0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of <em>in vitro </em>fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).</p>

Glutathione S-transferases Mu 3 (GSTM3) is an essential antioxidant enzyme whose presence in sperm has recently been related to sperm cryotolerance, quality and fertility. However, its role in seminal plasma (SP) as a predictor of the same sperm parameters has never been investigated. Herein, cell biology and proteomic approaches were performed to explore the presence, origin and role of SP-GSTM3 as a sperm quality and in vivo fertility biomarker. GSTM3 in SP was quantified using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit specific for Sus scrofa, whereas the presence of GSTM3 in testis, epididymis and accessory sex glands was assessed through immunoblotting analysis. Sperm quality and functionality parameters were evaluated in semen samples at 0 and 72 h of liquid-storage, whereas fertility parameters were recorded over a 12-months as farrowing rate and litter size. The presence and concentration of GSTM3 in SP was established for the first time in mammalian species, predominantly synthesized in the epididymis. The present study also evidenced a relationship between SP-GSTM3 and sperm morphology and suggested it is involved in epididymal maturation rather than in ejaculated sperm physiology. Finally, the data reported herein ruled out the role of this antioxidant enzyme as a quality and in vivo fertility biomarker of pig sperm ; The authors acknowledge the support from the Ministry of Science and Innovation (Spain), Ministry of Economy and Competitiveness (Spain) and FEDER funds (EU) (RYC-2014-15581, AGL2015-69738-R, AGL2017-88329-R, FJCI-2017-31689 and FPU18/00666); Seneca Foundation Murcia (Spain, 19892/GERM-15); and Regional Government of Catalonia, Spain (2017-SGR-1229)

Aquaporins (AQPs) are transmembrane channels with permeability to water and small solutes that can be classified according to their structure and permeability into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs. In boar spermatozoa, AQPs are related to osmoregulation and play a critical role in maturation and motility activation. In addition, their levels differ between ejaculates with good and poor cryotolerance (GFE and PFE, respectively). The aim of this work was to elucidate whether the involvement of AQPs in the sperm response to cryopreservation relies on the intrinsic freezability of the ejaculate. With this purpose, two different molecules: phloretin (PHL) and 1,3-propanediol (PDO), were used to inhibit sperm AQPs in GFE and PFE. Boar sperm samples were treated with three different concentrations of each inhibitor prior to cryopreservation, and sperm quality and functionality parameters were evaluated in fresh samples and after 30 and 240 min of thawing. Ejaculates were classified as GFE or PFE, according to their post-thaw sperm viability and motility. While the presence of PHL caused a decrease in sperm quality and function compared to the control, samples treated with PDO exhibited better quality and function parameters than the control. In addition, the effects of both inhibitors were more apparent in GFE than in PFE. In conclusion, AQP inhibition has more notable consequences in GFE than in PFE, which can be related to the difference in relative levels of AQPs between these two groups of samples ; The authors acknowledge the support from the European Commission (H2020-MSCA-IF-79212), the Ministry of Science, Innovation and Universities, Spain (grant nos. RYC-2014-15581, AGL2017-88329-R, FJCI-2017-31689, and PRE2018-083488), and the Regional Government of Catalonia, Spain (2017-SGR-1229)

The objective of present study is to identify the adverse effects of cyclosporine A drug and the protective role of garlic in cytotoxicity test, including sperms deformation and histological changes in testes tissues of male albino rats. The study recorded significant increase p≥0.05 in sperms head and tail deformation rates in groups that were administrated cyclosporine and cyclosporine + garlic for two weeks compared with control groups which were administrated olive oil and garlic. The study reported significant decrease p≥0.05 in sperms head and tail deformation rates in the group that was administrated cyclosporine + garlic compared with group that was administrated cyclosporine only. In addition, the study also indicated histological changes in testes tissues including degenerative changes represented by depletion of the spermatogonial cells (some seminiferous tubules appeared with one or two layers of spermatogonia) in the group that was administrated cyclosporine compared with the control group which was administrated olive oil. The study reported slight histological changes in testes tissues from normal testes architecture, somniferous tubules with maturation of spermatogony and sperms inside the lumen in group that was administrated cyclosporine + garlic for two weeks compared with the control group which was administrated olive oil and garlic. Finally, the findings showed reduction in histological changes of testes tissues in group which was administrated cyclosporine + garlic for two weeks compared with group which was administrated cyclosporine only.