Study of pufferfish (Takifugu niphobles) sperm: development of methods for short-term storage, effects of different activation media and role of intracellular changes in Ca2+ and K+ in the initiation of sperm motility

[EN] The first goal of this study was the development of a short-term storage method for pufferfish (Takifugu niphobles) sperm. In this respect, the best results were obtained by diluting the sperm in a seminal-like solution and keeping it in a Petri dish in chilled storage (4 degrees C). This method was successful in preserving sperm quality parameters without resulting in differences in fresh sperm for a relatively long-term period (7 days), for use in aquaculture matters. The addition of bovine serum albumin (BSA) to the medium did not improve the results. On the other hand, both the osmolality and the ion composition of the media are essential factors which can modulate the sperm motility parameters. The osmolality of the activating medium was the most important factor in triggering pufferfish sperm motility, and osmolalities of 750-825 mOsm/kg were necessary to initiate this process, demonstrating that it appears to be a dose-independent mechanism. Regarding the ion composition of the activation media, this study has shown that despite the spermatozoa being able to initiate movement without any ion in the activation medium, the absence of ions can negatively affect the kinetic parameters of the spermatozoa. Finally, in natural conditions (seawater), the activation of sperm motility generates intracellular increases in Ca2+ and K+, suggesting that these ions play an essential role in the activation mechanism of pufferfish sperm. (C) 2013 Elsevier B.V. All rights reserved.This study was funded by the Spanish Ministry of Science and Innovation (MICINN; AGL2010-16009). Victor Gallego has a predoctoral grant (MICINN; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) from the Spanish Personnel Research Training Programme to carry out this study in the Misaki Marine Biological Station (Miura, Japan).Gallego Albiach, V.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Yoshida, M. (2013). Study of pufferfish (Takifugu niphobles) sperm: development of methods for short-term storage, effects of different activation media and role of intracellular changes in Ca2+ and K+ in the initiation of sperm motility. Aquaculture. 414:82-91. https://doi.org/10.1016/j.aquaculture.2013.07.046S829141

Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results

[EN] Although it is widely accepted that osmolality and ion fluxes are the main factors triggering sperm motility in fish, a complex universal mechanism for sperm motility activation does not exist in fish, and studies of marine fish species are even more scarce. Therefore, the main goal of this study was to estimate the intracellular variations in the main ions involved in sperm activation for the first time in European eel, in order to provide additional new data about this little-known process. It was observed that levels of intracellular Ca2+ and K+ sperm ions increased significantly 30 s after the hyperosmotic shock compared to baseline levels, and remained at this level until 120 s post-activation. In contrast, the intracellular pH remained constant during the first 30 s, and decreased gradually at 60 and 120 s post-activation. Our data agree with the current main theory for explaining motility activation in marine fish, in which internal fluctuations of Ca2+ and K+ seem to participate in sperm activation. In addition, fluorescent images showed that both Ca2+ and K+ were concentrated in the apical area of the sperm head, which corresponds to the location of the eel sperm mitochondria, suggesting this organelle plays an important role in sperm motility activation. (C) 2013 Elsevier B.V. All rights reserved.Funded from the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology", grant agreement no 245257 (Pro-Eel) and the Spanish Ministry of Science and Innovation (MICINN; AGL2010-16009). Victor Gallego has a predoctoral grant (MICINN; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) of the Spanish Personnel Research Training Programme to carry out this study in the Universidad de Leon (Leon, Spain). Ilaria Mazzeo had a predoctoral grant from GVA. David S. Penaranda has a contract co-financed by MICINN and UPV (PTA2011-4948-I). F. Martinez-Pastor was supported by the Ramon y Cajal program (RYC-2008-02560, MICINN).Gallego Albiach, V.; Martínez Pastor, F.; Mazzeo, I.; Peñaranda, D.; Herraez, P.; Asturiano Nemesio, JF.; Pérez Igualada, LM. (2014). Intracellular changes in Ca2+, K+ and pH after sperm motility activation in the European eel (Anguilla anguilla): Preliminary results. Aquaculture. 418:155-158. https://doi.org/10.1016/j.aquaculture.2013.10.022S15515841

The role of the microbiome in ovarian cancer: mechanistic insights into oncobiosis and to bacterial metabolite signaling

Ovarian cancer is characterized by dysbiosis, referred to as oncobiosis in neoplastic diseases. In ovarian cancer, oncobiosis was identified in numerous compartments, including the tumor tissue itself, the upper and lower female genital tract, serum, peritoneum, and the intestines. Colonization was linked to Gram-negative bacteria with high inflammatory potential. Local inflammation probably participates in the initiation and continuation of carcinogenesis. Furthermore, local bacterial colonies in the peritoneum may facilitate metastasis formation in ovarian cancer. Vaginal infections (e.g. Neisseria gonorrhoeae or Chlamydia trachomatis) increase the risk of developing ovarian cancer. Bacterial metabolites, produced by the healthy eubiome or the oncobiome, may exert autocrine, paracrine, and hormonelike effects, as was evidenced in breast cancer or pancreas adenocarcinoma. We discuss the possible involvement of lipopolysaccharides, lysophosphatides and tryptophan metabolites, as well as, short-chain fatty acids, secondary bile acids and polyamines in the carcinogenesis of ovarian cancer. We discuss the applicability of nutrients, antibiotics, and probiotics to harness the microbiome and support ovarian cancer therapy. The oncobiome and the most likely bacterial metabolites play vital roles in mediating the effectiveness of chemotherapy. Finally, we discuss the potential of oncobiotic changes as biomarkers for the diagnosis of ovarian cancer and microbial metabolites as possible adjuvant agents in therapy

Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles)

[EN] The use of high quality gametes from both males and females during in vitro fertilization (IVF) trials is an essential step in order to achieve high fertilization and hatching rates. Although aquaculture hatcheries have focused more on egg rather than spermatozoa quality, some studies have demonstrated that sperm quantity and quality have a great influence both on fertilization/hatching success and the subsequent development of the embryo and larvae. In this study we have demonstrated that sperm/egg ratio and sperm quality are factors strongly related to each other in the pufferfish (Takifugu niphobles). Our results suggest that both factors should be taken into account as unique interrelated elements, making possible to obtain high fertilization rates using a successful combination of small amount of high quality sperm or high amount of low quality sperm. In addition, coefficients of correlation and determination among all the sperm motion parameters provided by a CASA system and fertilization/hatching rates were estimated for the first time in a marine species. Positive significant correlations were found in some parameters such as total and progressive motility (0.68 and 0.7 respectively). However, curvilinear velocity (VCL), straight line velocity (VSL) and average velocity (VAP) showed the highest coefficients of correlation (0.82, 0.8, and 0.81, respectively). In this respect, spermatozoa velocity appears to be a key factor in the fertilization process, especially when the number of spermatozoa per egg is limited in the aqueous environment. (C) 2013 Elsevier B.V. All rights reserved.Funded by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2010-16009). Victor Gallego has a predoctoral grant (MINECO; BES-2009-020310) and has been granted a fellowship (EEBB-I-12-05858) of the MINECO's Spanish Personnel Research Training Programme to carry out this research in the Misaki Marine Biological Station (Miura, Japan). We would like to thank to Dr. Kurokawa for the help and knowledge supplied during this study.Gallego Albiach, V.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Yoshida, M. (2013). Relationship between spermatozoa motility parameters, sperm/egg ratio, and fertilization and hatching rates in pufferfish (Takifugu niphobles). Aquaculture. 416:238-243. https://doi.org/10.1016/j.aquaculture.2013.08.035S23824341

Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux

Alterations in juvenile diploid and triploid African catfish skin gelatin yield and amino acid composition: effects of chlorpyrifos and butachlor exposures

Skin is a major by-product of the fisheries and aquaculture industries and is a valuable source of gelatin. This study examined the effect of triploidization on gelatin yield and proximate composition of the skin of African catfish (Clarias gariepinus). We further investigated the effects of two commonly used pesticides , chlorpyrifos (CPF) and butachlor (BUC), on the skin gelatin yield and amino acid composition in juvenile full-sibling diploid and triploid African catfish. In two separate experiments, diploid and triploid C. gariepinus were exposed for 21 days to graded CPF [mean measured: 10, 16, or 31 mg/L] or BUC concentrations [Mean measured: 22, 44, or 60 mg/L]. No differences in skin gelatin yield, amino acid or proximate compositions were observed between diploid and triploid control groups. None of the pesticide treatments affected the measured parameters in diploid fish. In triploids, however, gelatin yield was affected by CPF treatments while amino acid composition remained unchanged. Butachlor treatments did not alter any of the measured variables in triploid fish. To our knowledge, this study is the first to investigate changes in the skin gelatin yield and amino acid composition in any animal as a response to polyploidization and/or contaminant exposure

The Use of Anti-VDAC2 Antibody for the Combined Assessment of Human Sperm Acrosome Integrity and Ionophore A23187-Induced Acrosome Reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca2+ increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca2+ transmembrane transport

Ca2+ Extrusion by NCX Is Compromised in Olfactory Sensory Neurons of OMP−/− Mice

The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown.We used intact olfactory epithelium obtained from WT and OMP(-/-) mice to monitor the Ca(2+) dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca(2+) channels, or Ca(2+) stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca(2+)-homeostasis in these neurons by influencing the activity of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca(2+) elevation by stimulating the reverse mode of NCX in both WT and OMP(-/-) mice. The resulting Ca(2+) responses indicate that OMP facilitates NCX activity and allows rapid Ca(2+) extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM).Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions