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

Sperm motility parameters and spermatozoa morphometric characterization in marine species: a study of swimmer and sessile species

[EN] The biodiversity of marine ecosystems is diverse and a high number of species coexist side by side. However, despite the fact that most of these species share a common fertilization strategy, a high variability in terms of the size, shape, and motion of spermatozoa can be found. In this study, we have analyzed both the sperm motion parameters and the spermatozoa morphometric features of two swimmer (pufferfish and European eel) and two sessile (sea urchin and ascidian) marine species. The most important differences in the sperm motion parameters were registered in the swimming period. Sessile species sperm displayed notably higher values than swimmer species sperm. In addition, the sperm motilities and velocities of the swimmer species decreased sharply once the sperm was activated, whereas the sessile species were able to maintain their initial values for a long time. These results are linked directly to the species-specific lifestyles. Although sessile organisms, which show limited or no movement, need sperm with a capacity to swim for long distances to find the oocytes, swimmer organisms can move toward the female and release gametes near it, and therefore the spermatozoa does not need to swim for such a long time. At the same time, sperm morphology is related to sperm motion parameters, and in this study an in-depth morphometric analysis of ascidian, sea urchin, and pufferfish spermatozoa, using computer-assisted sperm analysis software, has been carried out for the first time. A huge variability in shapes, sizes, and structures of the studied species was found using electron microscopy. (C) 2014 Elsevier Inc. 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) of the Spanish Personnel Research Training Program to carry out this research in the Misaki Marine Biological Station (Miura, Japan).Gallego Albiach, V.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Yoshida, M. (2014). Sperm motility parameters and spermatozoa morphometric characterization in marine species: a study of swimmer and sessile species. Theriogenology. 82(5):668-676. https://doi.org/10.1016/j.theriogenology.2014.05.026S66867682

GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation

Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software

[EN] The development of powerful computer-assisted sperm analysis software has made kinetic studies of spermatozoa possible. This system has been used and validated for several species, but some technical questions have emerged regarding fish sample evaluations (i.e., frame rate, sperm dilution, chamber model, time of analysis, magnification lens, etc.). In the present study, we have evaluated the effects of different procedural and biological settings with the aim to correctly measure sperm quality parameters of the European eel. The use of different chambers did not affect the sperm motility parameters. However, regarding lens magnification, 10x was the most accurate lens, showing the least variation in the acquired data. Similarly, the frame rate setting resulted in a dramatic effect in some sperm kinetic parameters, primarily in terms of curvilinear velocity; we therefore recommend using the camera's highest available frame rate setting. Finally, the reduction in sperm motility over postactivation times suggests that sperm analysis should be performed within the first 60 seconds after activation of the European eel sperm. In conclusion, some protocol variables of sperm analysis by computer-assisted sperm analysis software can affect the measurement of eel sperm quality parameters, and should be considered before directly comparing results obtained by different laboratories. Moreover, because marine fish species show relatively similar features of sperm kinetic parameters, these results could be considered in the evaluation of the motility of sperm from other fish species. (C) 2013 Elsevier Inc. All rights reserved.This study was funded from the European Community's 7th Framework Programme under the Theme 2 "Food, Agriculture and Fisheries, and Biotechnology," grant agreement 245257 (Pro-Eel), and Generalitat Valenciana (ACOMP/2011/229). D.S.P. and P.C.F.C. have postdoctoral grants from UPV (CE-01-10) and PAC-EMBRAPA, respectively. I.M. and V.G. have predoctoral grants from Generalitat Valenciana and Spanish MICINN, respectively. The authors thank the Proiser R&D, S.L. team, who performed the task of fractioning the original sequences for the experiment described in section 3.2.Gallego Albiach, V.; Carneiro, PCF.; Mazzeo, I.; Vilchez Olivencia, MC.; Peñaranda, D.; Soler, C.; Pérez Igualada, LM.... (2013). Standardization of European eel (Anguilla anguilla) sperm motility evaluation by CASA software. Theriogenology. 79(7):1034-1040. https://doi.org/10.1016/j.theriogenology.2013.01.019S1034104079

Autonomic neuropathy predisposes to rosiglitazone-induced vascular leakage in insulin-treated patients with type 2 diabetes: a randomised, controlled trial on thiazolidinedione-induced vascular leakage

Contains fulltext : 88447.pdf (publisher's version ) (Closed access)AIMS/HYPOTHESIS: The mechanism of fluid-related complications caused by thiazolidinedione derivatives is unclear. One potential mechanism is thiazolidinedione-induced arterial vasodilatation, which results in vascular leakage and a fall in blood pressure, normally counterbalanced by sympathetic activation and subsequent renal fluid retention. We hypothesised that thiazolidinedione-induced vascular leakage will be particularly prominent in patients with autonomic neuropathy. METHODS: We conducted a randomised, double-blind, placebo-controlled, parallel study in 40 patients with type 2 diabetes on insulin treatment recruited from a university medical centre. The randomisation was performed by a central office using a randomisation schedule. Both treatment groups, placebo (n = 21) and rosiglitazone (n = 19), were stratified for sex and level of autonomic neuropathy as assessed by Ewing score (or=2.5). We investigated the effects of 16 weeks of treatment with rosiglitazone 4 mg twice daily on vascular leakage (transcapillary escape rate of albumin, TERalb), body weight, extracellular volume and plasma volume. RESULTS: Thirty-nine patients were included in the analysis. In patients with high Ewing scores (n = 16), rosiglitazone increased TERalb significantly (DeltaTERalb: rosiglitazone +2.43 +/- 0.45%/h, placebo -0.11 +/- 0.15%/h, p = 0.002), while rosiglitazone had no effect in the patients with low Ewing scores (n = 23). Rosiglitazone-induced increases in TERalb and Ewing score at baseline were correlated (r = 0.65, p = 0.02). There was no correlation between Ewing score and rosiglitazone-induced changes in fluid variables. One subject was withdrawn from the study because of atrial fibrillation. CONCLUSIONS/INTERPRETATION: Rosiglitazone may increase vascular leakage in insulin-treated patients with type 2 diabetes with autonomic neuropathy. Autonomic neuropathy did not exaggerate rosiglitazone-induced fluid retention. Therefore, autonomic neuropathy should be considered as a risk factor for thiazolidinedione-induced oedema, not for thiazolidinedione-induced fluid retention. TRIAL REGISTRATION: ClinicalTrials.gov NCT00422955. FUNDING: GlaxoSmithKline.1 september 201

Hysterectomy at a Canadian tertiary care facility: results of a one year retrospective review

BACKGROUND: The purpose of this study was to investigate the indications for and approach to hysterectomy at Kingston General Hospital (KGH), a teaching hospital affiliated with Queen's University at Kingston, Ontario. In particular, in light of current literature and government standards suggesting the superiority of vaginal versus abdominal approaches and a high number of concurrent oophorectomies, the aim was to examine the circumstances in which concurrent oophorectomies were performed and to compare abdominal and vaginal hysterectomy outcomes. METHODS: A retrospective chart audit of 372 consecutive hysterectomies performed in 2001 was completed. Data regarding patient characteristics, process of care and outcomes were collected. Data were analyzed using descriptive statistics, t-tests and linear and logistic regression. RESULTS: Average age was 48.5 years, mean body mass index (BMI) was 28.6, the mean length of stay (LOS) was 5.2 days using an abdominal approach and 3.0 days using a vaginal approach without laparoscopy. 14% of hysterectomies were performed vaginally, 5.9% were laparoscopically assisted vaginal hysterectomies and the rest were abdominal hysterectomies. The most common indication was dysfunctional or abnormal uterine bleeding (37%). The average age of those that had an oophorectomy (removal of both ovaries) was 50.8 years versus 44.3 years for those that did not (p < .05). Factors associated with LOS included surgical approach, age and the number of concurrent procedures. CONCLUSIONS: A significant reduction in LOS was found using the vaginal approach. Both the patient and the health care system may benefit from the tendency towards an increased use of vaginal hysterectomies. The audit process demonstrated the usefulness of an on-going review mechanism to examine trends associated with common surgical procedures

Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast

BACKGROUND: Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain. PRINCIPAL FINDINGS: Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast. CONCLUSIONS: The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity