Extracellular vesicles (EVs) are important effectors in cell-to-cell communication. Their cargo – including RNAs, lipids and proteins – is also provided to spermatozoa (SPZ) during their maturation, thus conferring them a new molecular guise. Obesity is an alarming health concern of modern society reaching epidemic dimensions. It is associated with an increased prevalence of male infertility, linked to a deregulated apoptosis of testicular germ cells, related to endoplasmic reticulum (ER) stress and, therefore, responsible for the alteration in sperm protein content. This affects sperm maturation and fertilization. To address this societal need an urgent development of innovative cell-based strategies is required, with a coordinated multi-tasking approach that investigates the role of EVs, which may supply novel insights into the mechanism by which obesity affects male fertility. In this frame, the major challenge of this project is the identification of the main mechanisms underlying the complex interplay linking EVs-SPZ communication and obesity-male infertility by using transcriptomic, proteomic and sphingolipidomic approaches. The target of these studies will be SPZ, collected from obese subjects and from the epididymis of high diet feed (HDF) mice. Seminal fluid will be also evaluated as a source of EVs of various origins (mainly epididymosomes and prostasomes). Furthermore, we will explore a new class of non-coding RNAs (ncRNAs) as possible regulators of sperm quality, affected by unhealthy diet habits and obesity: the circular RNAs (circRNAs), physically and functionally interacting with microRNAs (miRNAs), thus constituting a ceRNET (circRNA-associated- competitive endogenous RNA network). CeRNET associated with ER stress, apoptosis and infertility will be validated in both experimental models. Finally, the proteomic and sphingolipidomic profiles of EVs will be dissected in order to identify proteins and bioactive sphingolipids shuttled to SPZ and displaying altered profiles in both obese infertile patients and the HDF-mouse model. Moreover, caput/cauda epithelial murine epididymal cells will be used to demonstrate the role of sphingolipid signalling in epididymosome sorting of the identified ncRNAs and proteins..

The project is funded by European Union – Next-GenerationEU – National Recovery and Resilience PLAN (PNRR) – Mission 4, Component 2, Investiment 1.1 Fondo per il Programma Nazionale di Ricerca e Progetti di Rilevante Interesse Nazionale (PRIN). Project N. 20223H8LFM_001