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

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

A systematic analysis of the memory term in coarse-grained models: The case of the Markovian approximation

Copyright © The Author(s), 2022. The systematic development of coarse-grained (CG) models via the Mori–Zwanzig projector operator formalism requires the explicit description of a deterministic drift term, a dissipative memory term and a random fluctuation term. The memory and fluctuating terms are related by the fluctuation–dissipation relation and are more challenging to sample and describe than the drift term due to complex dependence on space and time. This work proposes a rational basis for a Markovian data-driven approach to approximating the memory and fluctuating terms. We assumed a functional form for the memory kernel and under broad regularity hypothesis, we derived bounds for the error committed in replacing the original term with an approximation obtained by its asymptotic expansions. These error bounds depend on the characteristic time scale of the atomistic model, representing the decay of the autocorrelation function of the fluctuating force; and the characteristic time scale of the CG model, representing the decay of the autocorrelation function of the momenta of the beads. Using appropriate parameters to describe these time scales, we provide a quantitative meaning to the observation that the Markovian approximation improves as they separate. We then proceed to show how the leading-order term of such expansion can be identified with the Markovian approximation usually considered in the CG theory. We also show that, while the error of the approximation involving time can be controlled, the Markovian term usually considered in CG simulations may exhibit significant spatial variation. It follows that assuming a spatially constant memory term is an uncontrolled approximation which should be carefully checked. We complement our analysis with an application to the estimation of the memory in the CG model of a one-dimensional Lennard–Jones chain with different masses and interactions, showing that even for such a simple case, a non-negligible spatial dependence for the memory term exists.Leverhulme Trust through Early Career Fellowship ECF-2016-52