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

“In medio stat virtus”: Insights into hybrid E/M phenotype attitudes

Epithelial-mesenchymal plasticity (EMP) refers to the ability of cells to dynamically interconvert between epithelial (E) and mesenchymal (M) phenotypes, thus generating an array of hybrid E/M intermediates with mixed E and M features. Recent findings have demonstrated how these hybrid E/M rather than fully M cells play key roles in most of physiological and pathological processes involving EMT. To this regard, the onset of hybrid E/M state coincides with the highest stemness gene expression and is involved in differentiation of either normal and cancer stem cells. Moreover, hybrid E/M cells are responsible for wound healing and create a favorable immunosuppressive environment for tissue regeneration. Nevertheless, hybrid state is responsible of metastatic process and of the increasing of survival, apoptosis and therapy resistance in cancer cells. The present review aims to describe the main features and the emerging concepts regulating EMP and the formation of E/M hybrid intermediates by describing differences and similarities between cancer and normal hybrid stem cells. In particular, the comprehension of hybrid E/M cells biology will surely advance our understanding of their features and how they could be exploited to improve tissue regeneration and repair

P-030 ACE2 receptor and its isoform short-ACE2 are expressed on human spermatozoa

STUDY QUESTION: Do human spermatozoa express angiotensin-converting enzyme 2 (ACE2) receptor? What would be its localization? SUMMARY ANSWER: Human spermatozoa express uniformly ACE2 on the sperm head and the flagellum. Moreover, the short-ACE2 isoform is concentrated on the post-acrosomal region and midpiece. WHAT IS KNOWN ALREADY: The Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) infection is generating important concerns regarding not only the possible consequences on the respiratory system, but also on other organs, including the reproductive system. ACE2 is considered the main point of entry for the SARS-CoV-2 within the cells through the binding with the spike protein on the virus surface. Furthermore, ACE2 is expressed in human testes cells including Leydig cells, Sertoli cells and spermatogonia. However, to date, the expression and location of ACE2 in mature human spermatozoa has not been investigated yet. STUDY DESIGN, SIZE, DURATION: This was an in vitro study for the evaluation of the expression and immune-localization of full-length ACE2 and its isoform, short-ACE2, in human spermatozoa. Thirthyfour non-immunized healthy normozoospermic volunteers were enrolled in the study. The study was conducted from May to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples were collected by masturbation from non-immunized healthy normozoospermic voluntaries. Motile sperm suspensions were obtained by swim-up procedure. The expression of ACE2 was assessed by Western-blot analysis, while the immune-localization of ACE2 was evaluated by immune-cytochemical analysis under confocal microscopy. Flow-cytometry experiments were also performed to assess the surface protein expression on a large number of cells. MAIN RESULTS AND THE ROLE OF CHANCE: The Western-blot analysis of sperm extracts demonstrated two specific bands, one of approximately 120 KDa, corresponding to the glycosylated full-length ACE2, and a second one of approximately 52 KDa, the molecular weight of the protein recently termed short-ACE2. The immune-cytochemical analysis showed a uniformly localization of full-length ACE2 along both the sperm head and the flagellum, whereas the short isoform was preferentially located in the post-acrosomal region of the sperm head and the midpiece. At the flow cytometer, semen samples displayed a wide between-subject variability both in the percentage of ACE2-positive spermatozoa and the density of protein surface expression. LIMITATIONS, REASONS FOR CAUTION: Further studies are needed to determine whether short-ACE2 is a cleavage product from the full-length protein or if it is originated during spermatogenesis. Moreover, the role and the interaction of ACE2 with SARS-CoV-2 in human spermatozoa should be clarified to evaluate the possible impact of the virus on sperm biology. WIDER IMPLICATIONS OF THE FINDINGS: Since mature spermatozoa are transcriptionally silent and SARS-CoV-2 is an RNA virus, it is unlikely that the virus could affect sperm biology by replicating itself. Nevertheless, the potential effects related to modifications of the sperm membrane or interaction with other receptors or specific proteins cannot be ruled out. TRIAL REGISTRATION NUMBER: not applicabl

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

Tendon-like Electrospun PLGA Scaffolds with Optimized Physical Cues Induced Tenogenic Differentiation and Boosted Immunomodulatory Properties on Amniotic Epithelial Stem Cells.

Introduction: The advanced strategies in the field of Tissue Engineering might render possible overcoming the unsatisfactory results of conventional treatments to deal with tendinopathies. In this context, the design of tendon biomimetic electrospun scaffolds engineered with Amniotic Epithelial Stem Cells (AECs), which have shown a high teno-regenerative and immunomodulatory potential in tendon-defect models, can represent a promising solution for tendon regeneration. Methods: Poly(lactide-co-glycolic) acid (PLGA) scaffolds were fabricated using the electrospinning technique to mimic the native tendon biomechanics and extracellular matrix by optimizing: fiber alignment and diameter size (1.27 and 2.5 µm), and surface chemistry using the Cold Atmospheric Plasma (CAP) Technique. Moreover, the teno-inductive and immunomodulatory effects of these parameters on AECs have been also assessed. Results: The fabricated PLGA scaffolds with highly aligned fibers and small diameter size (1.27 µm) induced a stepwise tenogenic differentiation on AECs with an early epithelial-mesenchymal transition (EMT), followed by their tenogenic differentiation. Indeed, SCX, an early tendon marker, was significantly more efficiently translated into the downstream effector TNMD, a mature tendon marker. Moreover, 1.27 µm fiber diameter induced on AECs a higher expression of anti-inflammatory interleukin mRNAs (IL-4 and IL-10). The CAP treated PLGA scaffolds showed an improved cell adhesion and infiltration without altering their topological structure and teno-inductive properties. In fact, AECs engineered with CAP treated fibers, expressed in their cytoplasm TNMD. Moreover, CAP treatment did not alter the mechanical properties of PLGA scaffolds. Conclusions: The developed electrospun PLGA scaffolds with the optimized features represent an ideal tendon-like construct that could be applied in in-vivo models to evaluate their biosafety and teno-regenerative potential

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

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

Amniotic Epithelial Stem Cells Counteract Acidic Degradation By-Products of Electrospun PLGA Scaffold by Improving their Immunomodulatory Profile In Vitro

Electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds with highly aligned fibers (ha-PLGA) represent promising materials in the field of tendon tissue engineering (TE) due to their characteristics in mimicking fibrous extracellular matrix (ECM) of tendon native tissue. Among these properties, scaffold biodegradability must be controlled allowing its replacement by a neo-formed native tendon tissue in a controlled manner. In this study, ha-PLGA were subjected to hydrolytic degradation up to 20 weeks, under di-H2 O and PBS conditions according to ISO 10993-13:2010. These were then characterized for their physical, morphological, and mechanical features. In vitro cytotoxicity tests were conducted on ovine amniotic epithelial stem cells (oAECs), up to 7 days, to assess the effect of non-buffered and buffered PLGA by-products at different concentrations on cell viability and their stimuli on oAECs’ immunomodulatory properties. The ha-PLGA scaffolds degraded slowly as evidenced by a slight decrease in mass loss (14%) and average molecular weight (35%), with estimated degradation half-time of about 40 weeks under di-H2 O. The ultrastructure morphology of the scaffolds showed no significant fiber degradation even after 20 weeks, but alteration of fiber alignment was already evident at week 1. Moreover, mechanical properties decreased throughout the degradation times under wet as well as dry PBS conditions. The influence of acid degradation media on oAECs was dose-dependent, with a considerable effect at 7 days’ culture point. This effect was notably reduced by using buffered media. To a certain level, cells were able to compensate the generated inflammation-like microenvironment by upregulating IL-10 gene expression and favoring an anti-inflammatory rather than pro-inflammatory response. These in vitro results are essential to better understand the degradation behavior of ha-PLGA in vivo and the effect of their degradation by-products on affecting cell performance. Indeed, buffering the degradation milieu could represent a promising strategy to balance scaffold degradation. These findings give good hope with reference to the in vivo condition characterized by physiological buffering systems