Different approaches for assessing sperm function

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the \u201cone shot\u201c parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their sub-population distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways

EFFECTS OF RESVERATROL ON VITRIFIED PORCINE OOCYTES

Vitrified MII porcine oocytes are characterized by reduced developmental competence, associated with the activation of the apoptotic pathway. Resveratrol (R), a polyphenolic compound present in several vegetal sources, has been reported to exert, among all its other biological effects, an antiapoptotic one. The aim of this study was to determine the effects of R (2\u2009\ub5M) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation. R was added during IVM (A); 2\u2009h postwarming incubation (B); vitrification/warming and 2\u2009h postwarming incubation (C); all previous phases (D). Data on PS exteriorization showed, in each treated group, a significantly higher (P<0.05) percentage of live nonapoptotic oocytes as compared with CTR; moreover, the percentage of live apoptotic oocytes was significantly (P<0.05) lower in all R-treated groups relative to CTR. The results on caspase activation showed a tendency to an increase of viable oocytes with inactive caspases in B, C, and D, while a significant (P<0.05) increase in A compared to CTR was recorded. These data demonstrate that R supplementation in various phases of IVM and vitrification/warming procedure can modulate the apoptotic process, improving the resistance of porcine oocytes to cryopreservation-induced damage

The sperm mitochondria: clues and challenges

Sperm cells rely on different substrates to fulfil thei energy demand for different functions and diverse moments of their life. Species specific mechanism involve both energy substrate transport and their utilization: hexose transporters, a protein family of facilitative passive transporters of glucose and other hexose, have been identified in spermatozoa of different species and, within the species, their localization has been identified and, in some cases, linked to specific glycilitic enzyme presence. The catabolism of hexose sources for energy purposes has been studied in various species, and recent advances has been made in the knowledge of metabolic strategies of sperm cells. In particular, the importance of aerobic metabolism has been defined and described in horse, boar and even mouse spermatozoa; bull sperm cells demonstrate to have a good adaptability and capacity to switch between glycolysis and oxidative phosphorylation; finally, dog sperm cells have been demonstrated to have a great plasticity in energy metabolism management, being also able to activate the anabolic pathway of glycogen syntesis. In conclusion, the study of energy management and mitochondrial function in spermatozoa of different specie furnishes important base knowledge to define new media for preservation as well as newbases for reproductive biotechnologies

A Systematic Analysis of the Memory term in Coarse-Grained models: the case of the Markovian Approximation

The systematic development of Coarse-Grained (CG) models via the Mori-Zwanzig projector operator formalism requires the explicit description of several terms, including a deterministic drift term, a dissipative memory term and a random fluctuation term. In many applications, the memory and fluctuation terms are related by the fluctuation-dissipation relation and are, in general, more challenging to derive than the drift term. In this work we analyse an approximation of the memory term and propose a rational basis for a data-driven approach to an approximation of the memory and fluctuating terms which can be considered included in the class of the Markovian ones.Comment: 39 pages, 2 Figure

A polyphenol-rich extract from an oenological oak-derived tannin influences in vitro maturation of porcine oocytes

Tannins have been demonstrated to have antioxidant and various health benefit properties. The aim of this study was to determine the effect of an ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R \uae ) on female gamete using an in vitro model of pig oocyte maturation (IVM) and examining nuclear maturation, cytoplasmic maturation, intracellular GSH and ROS levels and cumulus cell steroidogenesis. To this aim, during IVM performed in medium either supplemented (IVM A) or not supplemented (IVM B) with cysteine and \u3b2-mercaptoethanol, TRE was added at different concentrations (0, 1, 5, 10, 20 \u3bcg/ml). The addition of TRE at all the concentration tested to either IVM A or IVM B, did not influence oocyte nuclear maturation. When IVM was performed in IVM A, no effect was induced on cytoplasmic maturation by TRE at the concentration of 1, 5 and 10 \u3bcg/ml, while TRE 20 \u3bcg/ml significantly reduced the penetration rate after IVF (p &lt; 0.05) and the blastocyst rate after parthenogenetic activation (p &lt; 0.01). Oocyte maturation in IVM B, compared to IVM A group, decreased GSH (p &lt; 0.001) and increased ROS (p &lt; 0.01) intracellular levels and in turn impaired oocyte cytoplasmic maturation reducing the ability to sustain male pronuclear formation after IVM (p &lt; 0.001) and the developmental competence after parthenogenetic activation (p &lt; 0.001). TRE supplementation to IVM B significantly reduced ROS production (5, 10, 20 \u3bcg/ml TRE) to levels similar to IVM A group, and increased GSH levels (10, 20 \u3bcg/ml TRE) compared to IVM B (p &lt; 0.05) without reaching those of IVM A group. TRE supplementation to IVM B at the concentrations of 1, 5 and 10 \u3bcg/ml significantly improved (p &lt; 0.001) oocyte cytoplasmic maturation enhancing the ability to sustain male pronuclear formation without reaching, however, IVM A group levels. TRE addition at all the concentration tested to both IVM A and IVM B, did not induce any effect on E2 and P4 secretion by cumulus cells suggesting that the biological effect of the ethanol extract is not exerted thought a modulation of cumulus cell steroidogenesis. In conclusion, TRE, thanks to its antioxidant activity, was partially able to reduce the negative effect of the absence of cysteine and \u3b2-mercaptoethanol in IVM B, while TRE at high concentration in IVM A was detrimental for oocyte cytoplasmic maturation underlying the importance of maintaining a balanced redox environment during oocyte maturation

Geological map, balanced and restored cross-sections, and 3D geological model of the Monte Fema area, Umbria-Marche Apennines (Italy)

The Mt. Fema area is located within the 1:50,000 scale Sheet 325-Visso of the CARG project, in the Umbria-Marche Apennines. Here, inherited pre-orogenic deformation and multi-layered mechanical stratigraphy affect mountain belt evolution and cause along-and across-strike changes in structural architecture. Further complexity is caused by post-orogenic extensional tectonics dissecting the fold and thrust belt. In this work, we combined classical field methodologies with digital mapping and drone surveys to produce a 1:10,000 geological map of the Mt. Fema area. The resulting map was integrated with a 10 m-cell size DEM in a 3D environment to construct four balanced cross-sections that were used to document structural style and stratigraphic variations. One section was restored to quantify the amount of deformation related to both Neogene orogenic shortening and multiple extensional phases affecting the area. Ultimately, we built a 3D geological model to investigate the subsurface geometrical arrangement of strata and faults of different generations, thus the overall structural architecture of the fold and thrust belt. According to our interpretation, the Mt. Fema thrust system is characterised by relatively limited displacement (cumulative dip separation ranging from-100 m to the north in Val di Tazza to-500 m to the south in Valnerina). Reactivation of inherited normal faults was likely precluded because of their unfavourable orientation with respect to W-dipping thrusts. Inherited basin structure and mechanical stratigraphy govern folding by buckling mechanism, which in turn controls the locus of thrust propagation. Normal faults dissect the crestal region of the Mt. Fema anticline. These structures do not show evidence of surface faulting during recent seismic sequences, although earthquake epicentres fall within the study area. Our work provides new insights into the 3D structural architecture, timing, and kinematics of a key sector of the Umbria-Marche Apennines, with implications for a better understanding of the role of structural inheritance and subsequent extensional tectonics in the evolution of fold and thrust belts

GPR17 receptor modulators and their therapeutic implications: review of recent patents

Introduction: The GPR17 receptor, phylogenetically related to both purinergic P2Y and CysLT receptors, is mainly expressed in the CNS and, in general, in organs that can typically undergo ischemic damage. This receptor is involved in various pathologies including stroke, brain and spinal cord trauma, multiple sclerosis and in all diseases characterized by neuronal and myelin dysfunction. Therefore, there is a strong needed to identify molecules capable of binding specifically to GPR17 receptors. Areas covered: The review provides a summary of patents, published between 2009 and 2018, on chemicals and biologics and their clinical use. In this work, information is&nbsp;reported about the representative structures and biological activity of recently developed GPR17 receptor ligands. Expert opinion: The GPR17 receptor is an enigmatic receptor and an interesting therapeutic target in a variety of brain disorders and demyelinating diseases such as multiple sclerosis, stroke, schizophrenia, and depression. The modulation of this receptor could also be potentially useful in obesity treatment. Unfortunately, so far, there are no compounds under investigation in clinical trials but many researchers and companies are investing in the discovery of future potential GPR17 receptor drugs

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 \u3bcm) and EGCG (10, 20 and 60 \u3bcm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 \u3bcm (but not of 60 \u3bcm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 \u3bcm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone