Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability-An In Vitro Study in a Swine Model.

Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.

ACE2 Receptor and Its Isoform Short-ACE2 Are Expressed on Human Spermatozoa.

Angiotensin-converting enzyme 2 (ACE2) is a protein widely expressed in numerous cell types, with different biological roles mainly related to the renin-angiotensin system. Recently, ACE2 has been in the spotlight due to its involvement in the SARS-CoV-2 entry into cells. There are no data available regarding the expression of ACE2 and its short-ACE2 isoform at the protein level on human spermatozoa. Here, protein expression was demonstrated by western blot and the percentage of sperm displaying surface ACE2 was assessed by flow cytometry. Immunocytochemistry assays showed that full-length ACE2 was mainly expressed in sperm midpiece, while short ACE2 was preferentially distributed on the equatorial and post-acrosomal region of the sperm head. To our knowledge, this is the first study demonstrating the expression of protein ACE2 on spermatozoa. Further studies are warranted to determine the role of ACE2 isoforms in male reproduction.

Role and Modulation of TRPV1 in Mammalian Spermatozoa: An Updated Review.

Based on the abundance of scientific publications, the polymodal sensor TRPV1 is known as one of the most studied proteins within the TRP channel family. This receptor has been found in numerous cell types from different species as well as in spermatozoa. The present review is focused on analyzing the role played by this important channel in the post-ejaculatory life of spermatozoa, where it has been described to be involved in events such as capacitation, acrosome reaction, calcium trafficking, sperm migration, and fertilization. By performing an exhaustive bibliographic search, this review gathers, for the first time, all the modulators of the TRPV1 function that, to our knowledge, were described to date in different species and cell types. Moreover, all those modulators with a relationship with the reproductive process, either found in the female tract, seminal plasma, or spermatozoa, are presented here. Since the sperm migration through the female reproductive tract is one of the most intriguing and less understood events of the fertilization process, in the present work, chemotaxis, thermotaxis, and rheotaxis guiding mechanisms and their relationship with TRPV1 receptor are deeply analyzed, hypothesizing its (in)direct participation during the sperm migration. Last, TRPV1 is presented as a pharmacological target, with a special focus on humans and some pathologies in mammals strictly related to the male reproductive system.

Two-Player Game in a Complex Landscape: 26S Proteasome, PKA, and Intracellular Calcium Concentration Modulate Mammalian Sperm Capacitation by Creating an Integrated Dialogue-A Computational Analysis.

Recent experimental findings suggest the involvement of the 26S proteasome, the main protease active in eukaryotic cells, in the process that leads mammalian sperm to become fully fertile, so-called capacitation. Unfortunately, its role in male gametes signaling is still far from being completely understood. For this reason, here, we realized a computational model, based on network theory, with the aim of rebuilding and exploring its signaling cascade. As a result, we found that the 26S proteasome is part of a signal transduction system that recognizes the bicarbonate ion as an input terminal and two intermediate layers of information processing. The first is under the control of the 26S proteasome and protein kinase A (PKA), which are strongly interconnected, while the latter depends on intracellular calcium concentrations. Both are active in modulating sperm function by influencing the protein phosphorylation pattern and then controlling several key events in sperm capacitation, such as membrane and cytoskeleton remodeling. Then, we found different clusters of molecules possibly involved in this pathway and connecting it to the immune system. In conclusion, this work adds a piece to the puzzle of protease and kinase crosstalk involved in the physiology of sperm cells.

Cyclin-CDK Complexes are Key Controllers of Capacitation-Dependent Actin Dynamics in Mammalian Spermatozoa.

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional maturation process to acquire the competence to fertilize the female egg. During this process, called capacitation, the actin cytoskeleton dramatically changes its organization. First, actin fibers polymerize, forming a network over the anterior part of the sperm cells head, and then it rapidly depolymerizes and disappears during the exocytosis of the acrosome content (the acrosome reaction (AR)). Here, we developed a computational model representing the actin dynamics (AD) process on mature spermatozoa. In particular, we represented all the molecular events known to be involved in AD as a network of nodes linked by edges (the interactions). After the network enrichment, using an online resource (STRING), we carried out the statistical analysis on its topology, identifying the controllers of the system and validating them in an experiment of targeted versus random attack to the network. Interestingly, among them, we found that cyclin-dependent kinase (cyclin-CDK) complexes are acting as stronger controllers. This finding is of great interest since it suggests the key role that cyclin-CDK complexes could play in controlling AD during sperm capacitation, leading us to propose a new and interesting non-genomic role for these molecules.

Autophagy is involved in granulosa cell death and follicular atresia in ewe ovaries.

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.

Edible insects and legumes exert an antioxidant effect on human colon mucosal cells stressed with 2,2'-azobis (2-amidinopropane)-dihydrochloride.

Introduction: Edible insects have been recognized as a more sustainable source of nutrients and bio-active compounds than animal-based products, in line with classical vegetable sources such as legumes. In this study, we assessed the antioxidant properties of four edible insects (silkworms, grasshoppers, mealworms and giant worms) and four legume seeds (lentils, chickpeas, Roveja peas and grass peas). Methods: After the aqueous extraction or in vitro simulated digestion process, selected products were assessed for: (i) in vitro antioxidant capacity through Ferric Reducing Antioxidant Power (FRAP) assay; (ii) the ability to reduce free radicals production induced by a pro-oxidant agent in cells of human colonic mucosa. Results: All the aqueous extracts and digesta of edible insects displayed significantly higher in vitro antioxidant activity than legumes. Moreover, edible insects at all tested concentrations were able to exert an antioxidant effect in the cellular model, while legumes were effective mainly at high concentrations. Discussion: Despite human trials are need to confirm and define these results in a physiological situation, here we suggest a role for edible insects in oxidative stress prevention. Since oxidative stress is strongly correlated with several intestinal pathologies, the results obtained could be interesting for the prevention and relief of the negative symptoms, offering new advantages to their already known ecological and nutritional properties.

An interactive analysis of the mouse oviductal miRNA profiles.

MicroRNAs are small non-coding molecules that control several cellular functions and act as negative post-transcriptional regulators of the mRNA. While their implication in several biological functions is already known, an important role as regulators of different physiological and pathological processes in fertilization and embryo development is currently emerging. Indeed, miRNAs have been found in the oviductal fluid packaged within the extracellular vesicles, which might act as natural nanoshuttles by transporting lipids, proteins, RNA molecules and miRNAs from the oviduct to the gametes or embryos. Here, an exhaustive bibliography search was carried out, followed by the construction of a computational model based on the networks theory in an attempt to recreate and elucidate the pathways potentially activated by the oviductal miRNA. The omics data published to date were gathered to create the Oviductal MiRNome, in which the miRNA target genes and their interactions are represented by using stringApp and the Network analyzer from Cytoscape 3.7.2. Then, the hyperlinked nodes were identified to investigate the pathways in which they are involved using the gene ontology enrichment analysis. To study the phenotypical effects after the removal of key genes on the reproductive system and embryo, knockout mouse lines for every protein-coding gene were investigated by using the International Mouse Phenotyping Consortium database. The creation of the Oviductal MiRNome revealed the presence of important genes and their interactions within the network. The functional enrichment analysis revealed that the hyperlinked nodes are involved in fundamental cellular functions, both structural and regulatory/signaling, suggesting their implication in fertilization and early embryo development. This fact was as well evidenced by the effects of the gene deletion in KO mice on the reproductive system and embryo development. The present study highlights the importance of studying the miRNA profiles and their enormous potential as tools to improve the assisted reproductive techniques currently used in human and animal reproduction.

Pre-Treatment of Swine Oviductal Epithelial Cells with Progesterone Increases the Sperm Fertilizing Ability in an IVF Model.

Mammalian spermatozoa are infertile immediately after ejaculation and need to undergo a functional modification, called capacitation, in order to acquire their fertilizing ability. Since oviductal epithelial cells (SOECs) and progesterone (P4) are two major modulators of capacitation, here we investigated their impact on sperm functionality by using an IVF swine model. To that, we treated SOECs with P4 at 10, 100, and 1000 ng/mL before the coincubation with spermatozoa, thus finding that P4 at 100 ng/mL does not interfere with the cytoskeleton dynamics nor the cells' doubling time, but it promotes the sperm capacitation by increasing the number of spermatozoa per polyspermic oocyte (p < 0.05). Moreover, we found that SOECs pre-treatment with P4 100 ng/mL is able to promote an increase in the sperm fertilizing ability, without needing the hormone addition at the time of fertilization. Our results are probably due to the downregulation in the expression of OVGP1, SPP1 and DMBT1 genes, confirming an increase in the dynamism of our system compared to the classic IVF protocols. The results obtained are intended to contribute to the development of more physiological and efficient IVF systems.

Graphene and Reproduction: A Love-Hate Relationship.

Since its discovery, graphene and its multiple derivatives have been extensively used in many fields and with different applications, even in biomedicine. Numerous efforts have been made to elucidate the potential toxicity derived from their use, giving rise to an adequate number of publications with varied results. On this basis, the study of the reproductive function constitutes a good tool to evaluate not only the toxic effects derived from the use of these materials directly on the individual, but also the potential toxicity passed on to the offspring. By providing a detailed scientometric analysis, the present review provides an updated overview gathering all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported to date from experiments performed in vivo and in vitro and in different animal species (from Archea to mammals). Special attention is given to the oxidized form of graphene, graphene oxide, which has been recently investigated for its ability to increase the in vitro fertilization outcomes. Thus, the potential use of graphene oxide against infertility is hypothesized here, probably by engineering the spermatozoa and thus manipulating them in a safer and more efficient way.

The impact of five years storage/biobanking at -80°C on mouse spermatozoa fertility, physiology, and function.

BACKGROUND: We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a -80°C freezer, maintaining their ability to fertilize mouse eggs. OBJECTIVES: The main objective here was to evaluate the effects of five years at -80°C compared to liquid nitrogen storage (LN2 , control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. MATERIALS AND METHODS: Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR-14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3-AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. RESULTS: Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at -80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2 . DISCUSSION: Long-term mouse sperm storage at -80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. CONCLUSION: It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a -80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.

Human Immune System Diseasome Networks and Female Oviductal Microenvironment: New Horizons to be Discovered.

Human hypofertility and infertility are two worldwide conditions experiencing nowadays an alarming increase due to a complex ensemble of events. The immune system has been suggested as one of the responsible for some of the etiopathogenic mechanisms involved in these conditions. To shed some light into the strong correlation between the reproductive and immune system, as can be inferred by the several and valuable manuscripts published to date, here we built a network using a useful bioinformatic tool (DisGeNET), in which the key genes involved in the sperm-oviduct interaction were linked. This constitutes an important event related with Human fertility since this interaction, and specially the spermatozoa, represents a not-self entity immunotolerated by the female. As a result, we discovered that some proteins involved in the sperm-oviduct interaction are implicated in several immune system diseases while, at the same time, some immune system diseases could interfere by using different pathways with the reproduction process. The data presented here could be of great importance to understand the involvement of the immune system in fertility reduction in Humans, setting the basis for potential immune therapeutic tools in the near future.

Progesterone induces sperm release from oviductal epithelial cells by modifying sperm proteomics, lipidomics and membrane fluidity.

The sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) -3 and -5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls.

Graphene Oxide Improves in vitro Fertilization in Mice With No Impact on Embryo Development and Preserves the Membrane Microdomains Architecture.

During the latest years, human infertility worsened all over the world and is nowadays reputed as a global public health issue. As a consequence, the adoption of Assisted Reproductive Technologies (ARTs) such as In Vitro Fertilization (IVF) is undergoing an impressive increase. In this context, one of the most promising strategies is the innovative adoption of extra-physiological materials for advanced sperm preparation methods. Here, by using a murine model, the addition of Graphene Oxide (GO) at a specific concentration has demonstrated to increase the spermatozoa fertilizing ability in an IVF assay, finding that 0.5 µg/ml GO addition to sperm suspensions before IVF is able to increase both the number of fertilized oocytes and embryos created with a healthy offspring given by Embryo Transplantation (ET). In addition, GO treatment has been found more effective than that carried out with methyl-ß-cyclodextrin, which represents the gold standard in promoting in vitro fertility of mice spermatozoa. Subsequent biochemical characterization of its interaction with male gametes has been additionally performed. As a result, it was found that GO exerts its positive effect by extracting cholesterol from membranes, without affecting the integrity of microdomains and thus preserving the sperm functions. In conclusion, GO improves IVF outcomes in vitro and in vivo, defining new perspectives for innovative strategies in the treatment of human infertility.

Graphene Oxide increases mammalian spermatozoa fertilizing ability by extracting cholesterol from their membranes and promoting capacitation.

Graphene Oxide (GO) is a widely used biomaterial with an amazing variety of applications in biology and medicine. Recently, we reported the ability of GO to improve the in vitro fertilization (IVF) outcomes in swine, a validated animal model with a high predictive value for human fertility. For that reason, here we characterized the mechanisms involved in this positive interaction by adopting an experimental approach combining biological methods (confocal microscopy analysis on single cell, flow cytometry on cell populations and co-incubation with epithelial oviductal cells), physical-chemical techniques (Differential Scanning Calorimetry and Thermogravimetric Analysis), and chemical methods (mass spectrometry and lipid measurement). As a result, we propose a model in which GO is able to extract cholesterol from the spermatozoa membrane without causing any detrimental effect. In this way, the cholesterol extraction promotes a change in membrane chemical-physical properties that could positively affect male gamete function, modulating sperm signalling function and increasing in this way the fertilizing potential, without losing the ability to physiologically interact with the female environment. In conclusion, these data seem to suggest new intriguing possibilities in engineering sperm membrane for improving assisted reproduction technologies outcomes, even in human medicine.

Aminopurvalanol A, a Potent, Selective, and Cell Permeable Inhibitor of Cyclins/Cdk Complexes, Causes the Reduction of in Vitro Fertilizing Ability of Boar Spermatozoa, by Negatively Affecting the Capacitation-Dependent Actin Polymerization.

The adoption of high-througput technologies demonstrated that in mature spermatozoa are present proteins that are thought to be not present or active in sperm cells, such as those involved in control of cell cycle. Here, by using an in silico approach based on the application of networks theory, we found that Cyclins/Cdk complexes could play a central role in signal transduction active during capacitation. Then, we tested this hypothesis in the vitro model. With this approach, spermatozoa were incubated under capacitating conditions in control conditions (CTRL) or in the presence of Aminopurvalanol A a potent, selective and cell permeable inhibitor of Cyclins/Cdk complexes at different concentrations (2, 10, and 20 µM). We found that this treatment caused dose-dependent inhibition of sperm fertilizing ability. We attribute this event to the loss of acrosome integrity due to the inhibition of physiological capacitation-dependent actin polymerization, rather than to a detrimental effect on membrane lipid remodeling or on other signaling pathways such as tubulin reorganization or MAPKs activation. In our opinion, these data could revamp the knowledge on biochemistry of sperm capacitation and could suggest new perspectives in studying male infertility.

Networks Models of Actin Dynamics during Spermatozoa Postejaculatory Life: A Comparison among Human-Made and Text Mining-Based Models.

Here we realized a networks-based model representing the process of actin remodelling that occurs during the acquisition of fertilizing ability of human spermatozoa (HumanMade_ActinSpermNetwork, HM_ASN). Then, we compared it with the networks provided by two different text mining tools: Agilent Literature Search (ALS) and PESCADOR. As a reference, we used the data from the online repository Kyoto Encyclopaedia of Genes and Genomes (KEGG), referred to the actin dynamics in a more general biological context. We found that HM_ALS and the networks from KEGG data shared the same scale-free topology following the Barabasi-Albert model, thus suggesting that the information is spread within the network quickly and efficiently. On the contrary, the networks obtained by ALS and PESCADOR have a scale-free hierarchical architecture, which implies a different pattern of information transmission. Also, the hubs identified within the networks are different: HM_ALS and KEGG networks contain as hubs several molecules known to be involved in actin signalling; ALS was unable to find other hubs than "actin," whereas PESCADOR gave some nonspecific result. This seems to suggest that the human-made information retrieval in the case of a specific event, such as actin dynamics in human spermatozoa, could be a reliable strategy.