Nanotechnology-Assisted Cell Tracking

The usefulness of nanoparticles (NPs) in the diagnostic and/or therapeutic sector is derived from their aptitude for navigating intra-and extracellular barriers successfully and to be spatiotemporally targeted. In this context, the optimization of NP delivery platforms is technologically related to the exploitation of the mechanisms involved in the NP–cell interaction. This review provides a detailed overview of the available technologies focusing on cell–NP interaction/detection by describing their applications in the fields of cancer and regenerative medicine. Specifically, a literature survey has been performed to analyze the key nanocarrier-impacting elements, such as NP typology and functionalization, the ability to tune cell interaction mechanisms under in vitro and in vivo conditions by framing, and at the same time, the imaging devices supporting NP delivery assessment, and consideration of their specificity and sensitivity. Although the large amount of literature information on the designs and applications of cell membrane-coated NPs has reached the extent at which it could be considered a mature branch of nanomedicine ready to be translated to the clinic, the technology applied to the biomimetic functionalization strategy of the design of NPs for directing cell labelling and intracellular retention appears less advanced. These approaches, if properly scaled up, will present diverse biomedical applications and make a positive impact on human health

P-440 Impact of electrospun scaffold topology on the performance of in-vitro Folliculogenesis applied to preantral ovine follicles

Study question How to improve in-vitro Folliculogenesis (ivF) protocols to address the enlarged demand of fertility preservation? Summary answer Tissue engineering-based approach opens new frontiers for ivF improving 3D-technologies addressed to support immature-ovarian-follicle-growth to obtain an increased number of competent oocytes enrolled in Assisted-Reproductive-Technology. What is known already ivF is a promising Assisted-Reproductive-Technology (ART) for preserving and restoring fertility. This technology potentially reproduces the early stages of folliculogenesis and oogenesis in-vitro allowing to move a large amount of oocyte on individual basis towards the validated protocol of in-vitro maturation/in-vitro fertilization (IVM/IVF). The current availability of biocompatible-supporting materials offers the challenging opportunity to mimic the native organ stroma in order to better reproduce the 3D environmental conditions leading to synergic follicles-oocyte development in-vitro with the aim to improve the performance of ivF in translational large sized mammal models. Study design, size, duration The present research aimed to compare preantral (PA) follicles culture on two different typologies of scaffolds fabricated using PCL(poly(epsilon caprolactone)), respectively made with patterned and randomly aligned fibers (PCL-Patterned/PCL-Randomic) with a standardized-single-follicle scaffold-free-method (3D-oil), widely validated on ovine model (Cecconi et al., 2004). The culture outcomes are compared analyzing follicle/oocyte growth, percentage of antrum differentiation and the incidence of meiotic competence, by exposing ivF growing oocytes to IVM protocol. Participants/materials, setting, methods PA follicles (mean size diameter: 250±4μm), mechanically isolated from slaughterhoused lamb ovaries, were individually cultured on electrospun PCL scaffolds (patterned vs randomic) or using the 3D-oil method. ivF were cultured alphaMEM-Fetal Bovine Serum free medium (5% Knockout Serum Replacement) supplemented with 4 IU/mL of equine Chorionic Gonadotropin (Di Berardino et al., 2021). At the end of ivF (14-days) the fully-grown oocytes isolated from early-antral follicles were tested on IVM. Main results and the role of chance PCL-Patterned electrospun scaffolds were able to strongly support a synergic oocyte and follicular growth. The 3D culture on Patterned electrospun scaffold supported the highest viability of follicles (87.5% vs 63% under 3D-oil conditions). On the contrary, the highest incidence of degenerated follicles was observed in cultures performed using PCL-Randomic materials (55 vs 37% vs 12.5% for PCL-Randomic vs 3D-oil vs PCL-Patterned, respectively; p <0.0004). The greatest follicle diameter increment (74.7±1 vs 70±0.4 vs 60.9±2%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, respectively p <0.0007) and rate of antrum differentiation (87.5% vs 45% and vs 63%, for PCL-Patterned vs 3D-oil vs PCL-Randomic, for both p <0.0001) were observed in PA ovine follicles cultured on PCL-Patterned scaffolds. Furthermore, PCL-Patterned electrospun scaffolds supported a complete functional development of the oocyte compartment. More in detail, the majority of fully grown oocytes isolated from early- antral follicles grown on PCL-Patterned materials reached the metaphase-II stage (MII 80%) at the end of IVM in comparison to the significant lower percentage in 3D-oil (MII 68%, p =0.04) and PCL-Randomic (MII 18%, p <0.0001) protocols, respectively. Limitations, reasons for caution - Wider implications of the findings Tissue engineering scaffold-based approach represents a valid strategy generating a multi-organ in-vitro system, where different compartments may cooperate generating the complexity of paracrine-mechanism controlling early-follicles outcomes. Scaffold topology is essential to control early-follicles development. Indeed, exclusively PCL-Patterned can preserve long-term follicle 3D-microarchitecture supporting in-vitro oogenesis up to a complete meiotic-competence-acquisition. Trial registration number not applicabl

26S PROTEASOME AND PKA MODULATE MAMMALIAN SPERM CAPACITATION BY CREATING AN INTEGRATED DIALOGUE: A COMPUTATIONAL ANALYSIS

Recent experimental evidence suggests 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 as an attempt to rebuild and explore 26S proteasome signaling cascade, aggregating all the molecular data available to date and realizing the Proteasome Interactome Network (PIN). Once obtained the network (i.e., a graph to represent the molecules as nodes and the interactions among them as links), we assessed its topology to infer important biological information. PIN is composed of 157 nodes, 248 links and it is characterized by a scale-free topology, following the Barabasi Albert model. In other words, it possesses a large amount of scarcely linked nodes and a small set of highly linked nodes, the hubs, which act as system controllers. This peculiar topology confers to the network relevant biological features: it is robust against random attacks, easily navigable and controllable and it is possible to infer new information from it. Indeed, the analysis of PIN showed that PKA and 26S proteasome were strongly interconnected and both were active in sperm signaling by influencing the protein phosphorylation pattern and then controlling several key events in sperm capacitation, such as membrane and cytoskeleton remodeling. In conclusion, the network model could explain many biological aspects of sperm physiology that are out of focus looking at the single molecular determinant, overcoming the reductionist approach which did not consider the complexity of molecules and their interactions. This could be helpful to identify potential diagnostic markers and therapeutic strategies concurring in explaining and approaching male infertility

Fractal analysis of microCT images of the oviduct during the estrous cycle

It is well known that the oviduct plays a key role in several events deeply related with reproduction, such as sperm storage and capacitation, gametes interactions, fertilization and early embryo development, among others. To better understand some of the interactions and process occurring withing this organ, the study of its morphological modifications is of primordial importance. To that, we adopted a microtomografy (MicroTC) modelling system and the fractal analysis that allow to explore the 3D oviductal functional anatomy, by using eight swine oviducts at different stages of the estrous cycle. MicroCT datasets were acquired by using the high-resolution 3D-imaging system Skyscan 1172G (Bruker, Kontich – Belgium). CT images were analyzed using plugin on ImageJ software (NIH, Bethesda, MD), a box-counting method was applied to calculate the Fractal dimension of the oviduct. Focusing our attention on the utero-tubal junction (involved in sperm selection) and the isthmo-ampullar junction (the fertilization site). We found that by using PCA analysis it was possible to clearly differentiate the oviduct at different cycle stage on the basis of their values for: Db for grid, lacunarity for grid, R2 for Db, Media Db, lacunarity, σ for D for Db, Max for D, Min for D. Lacunarity, Media and Max for Db have a greater influence on the analysis. The results showed that 2 principal components were associated whit the morphological changes. This information, is obtained by a fast nondestructive method, and could be very useful because this innovative approach enables the achievement a 3D model and suggest that using fractal analysis techniques can aid to better understand the modifications of oviduct anatomy that depends on the neuroendocrine axis. This innovative approach could be a start point to design 3D cell culture systems, that could be applied in human and animal assisted reproductive techniques, improving the IVF outcomes

Histology and carbohydrate histochemistry of the alimentary canal in the rainbow trout Oncorhinchus mykiss

A histological and histochemical analyses were carried out on the entire alimentary canal of the rainbow trout Oncorhynchus mykiss. In particular the oesophageal region showed presence of terminal β-D-galactose(1-3)-N- acetylgalactosamine and α-N-acetylgalactosamine. In the anterior and posterior regions of the stomach, lining epithelium and gastric pits exhibited the presence of β-gal and α-GalNAc. In addition sialoglycoconjugates having sialic acid-β-galactose(1-3)-N-acetylgalactosamine and sialic acid-N-acetylgalactosamine as terminal tri- and di-saccharides, were demonstrated. In proximal and distal intestine goblet cells showed the presence of sialoglyconjugates, having sialic acid-β-gal(1-3)-GalNAc and sialic acid-GalNAc as terminal sequences, belonging to N-linked chains. In the enterocytes of the entire intestine, terminal GlcNAc, α-Gal, α-fucose were found